Van de Poel Cloning Manual

VAN DE POEL 实验室克隆手册

DNA extraction

DNA 提取

gDNA extraction from Arabidopsis for cloning

用于克隆的拟南芥 gDNA 提取 Extraction buffer:

提取缓冲液：

200 mM Tris-HCl pH 7.5 250 mM NaCl 25 mM EDTA 0.5 % SDS

💡 注释 (Annotation):

Tris-HCl: 维持 pH 值稳定，保护 DNA 不被酸碱破坏。

NaCl: 提供离子环境，有助于维持 DNA 结构并帮助蛋白质沉淀。

EDTA: 螯合镁离子 (Mg²⁺)。由于 DNase（DNA酶）需要 Mg²⁺ 才能降解 DNA，EDTA 能有效抑制

DNase 活性，同时破坏细胞膜的稳定性。

SDS: 十二烷基硫酸钠，一种强效去污剂。用于裂解细胞膜和核膜，并使蛋白质变性，将其与 DNA 分离。

Protocol:

实验方案：

💡 注释: 液氮速冻可以瞬间停止所有酶的活性（防止内源性 DNase 降解 DNA），并使植物组织变脆， 易于研磨破碎。

💡 注释: 离心将细胞碎片（沉淀在底部）与含有溶解 DNA 的溶液（上清液）分离。

💡 注释: 异丙醇会改变溶液的介电常数，使 DNA 分子更容易相互聚集并从水中析出（沉淀）。低温有助于提高沉淀效率。

💡 注释: 70% 乙醇用于洗涤 DNA 沉淀。其中的水成分可以溶解共沉淀的盐分（盐分会抑制后续酶反应），而乙醇成分则保持 DNA 处于不溶解的沉淀状态。

💡 注释: 残留的乙醇会严重抑制后续 PCR 或酶切反应，必须挥发干净。但也不能过度干燥（变透明），否则 DNA 难以重新溶解。

Rough gDNA extraction from Arabidopsis, Marchantia or

Physcomitrium for quick genotyping

用于快速基因分型的拟南芥、地钱或小立碗藓粗糙 gDNA 提取 Solutions (both from sigmaaldrich.com):

溶液（均购自 sigmaaldrich.com）：

(Extract-N-Amp Direct PCR Extraction Solution)

(Extract-N-Amp 直接 PCR 提取液) Dilution solution D5688-12mL (Dilution Solution, Plant Direct PCR Sigma-Aldrich) 稀释液 D5688-12mL (植物直接 PCR 稀释液，Sigma-Aldrich)

Both solutions were originally intended to be used for the Extract-N-Amp Plant PCR kit (sigmaaldrich.com). However, a modified version of the manufacturer's protocol can be used to prep gDNA from different plant species for genotyping (up to 1000 bp fragments) and can be tested to be used for cloning (quality might not be good enough for difficult PCR amplifications or downstream cloning).

这两种溶液最初是用于 Extract-N-Amp 植物 PCR 试剂盒 (sigmaaldrich.com)。然而，可以使用制造商方案的修改版本来制备不同植物物种的 gDNA 用于基因分型（最高 1000 bp 片段），也可以测试用于克隆（但对于困难的 PCR 扩增或下游克隆，其质量可能不够好）。

Protocol:

实验方案：

Transfer a small piece of plant material into the tube (for Arabidopsis: small leaf from seedlings or a piece of a leaf from older plants; for Marchantia or Physcomitrium: small piece of plant material).

将一小块植物材料转移到管中（对于拟南芥：幼苗的小叶或老植株的叶片碎片；对于地钱或小立碗藓：一小块植物材料）。

💡 注释: 高温用于裂解细胞释放 DNA。

💡 注释: 稀释液用于中和提取液中的抑制成分，使环境适合 PCR 酶。

DNA synthesis

DNA 合成 Synthetic genes:

合成基因：

(If the prices change in the future, alternative companies could be: TwistBioscience or BioCat) （如果未来价格变动，替代公司可以是：TwistBioscience 或 BioCat）

Codon optimisation密码子优化

Primers are ordered as “Single-stranded DNA” in tubes, generally at a concentration of 25 nmole

DNA.

引物通常以“单链 DNA”的形式订购于管中，浓度通常为 25 nmole DNA。

Plasmid miniprep

质粒小量制备（Miniprep） Execute all steps at room temperature. We use the Thermo Scientific GeneJJET Plasmid Miniprep kit (K0502).

所有步骤均在室温下执行。我们使用 Thermo Scientific GeneJET 质粒小量制备试剂盒 (K0502)。

Before you start:

开始之前：

💡 注释: RNase A 用于降解细菌 RNA。如果不去除 RNA，它会干扰后续的酶切和电泳分析，使条带模糊。

Check if ethanol (96 %) is added to the Wash solution (in a ratio 1:1.75 or 35 mL EtOH per 20 mL

💡 注释: 裂解液（Lysis buffer，通常含 SDS/NaOH）在低温下容易析出 SDS 白色沉淀。如果不溶解， 裂解效率会大幅下降。

Protocol:

实验方案：

• To increase plasmid yield for low-copy numbers: take 2 or 3 x 2 mL culture (or spin down 10 mL culture in a falcon) and pool them after the resuspension step.
• 若要增加低拷贝数质粒的产量：取 2 或 3 管 2 mL 培养物（或在 Falcon 管中离心 10 mL 培养物），并在重悬步骤后将它们合并。

• If you have multiple pellets from the same culture, only use 250 uL Resuspension Solution once for all pellets.
• 如果你有来自同一培养物的多个沉淀，所有沉淀只使用一次 250 uL 重悬液。

💡 注释: 裂解液呈碱性且含 SDS，能破裂细胞并使 DNA 变性。切勿涡旋，否则会物理剪切细菌基因组

DNA，导致其断裂成小片段污染质粒提取物。

💡 注释: 中和液使 pH 值恢复中性。质粒 DNA 较小，能迅速复性（重新形成双螺旋），而基因组 DNA 较大，无法复性并与变性蛋白质、SDS 形成白色絮状沉淀。

💡 注释: 质粒 DNA 在高盐环境下特异性结合到硅胶膜上。

Add 500 uL Wash Solution to the GeneJET spin column.

向 GeneJET 离心柱中加入 500 uL 洗涤液（Wash Solution）。

💡 注释: 此步骤非常关键，用于去除残留的乙醇。残留乙醇会抑制洗脱和后续的所有酶反应（如 PCR、 酶切）。

💡 注释: 孵育可以让洗脱液充分渗透膜，溶解 DNA，提高产量。

• You can resuspend the flow-through containing the plasmid on the same spin column membrane and repeat steps the last two steps to get a slightly higher plasmid yield.
• 你可以将含有质粒的流穿液重新加到同一个离心柱膜上，并重复最后两个步骤，以获得稍高的质粒产量。

Freezer stocks

菌种保藏（甘油菌） Take 0.75 mL of overnight bacterial culture and pipet in an autoclaved 1.5 mL screw-cap tube.

取 0.75 mL 过夜细菌培养物，移液至高压灭菌的 1.5 mL 螺旋盖管中。

💡 注释: 甘油作为抗冻剂，防止冰晶形成刺破细菌细胞膜，从而在低温下保护细菌。

PCR on DNA template

Reaction Setup for Phusion Polymerase:

Phusion 聚合酶反应体系：

All components should be mixed and centrifuged prior to use. Assemble all reaction components on ice and quickly transfer the reactions to a thermocycler preheated to the denaturation temperature (98°C).

Component (组分) 20 µL Reaction 50 µL Reaction Final Concentratio

Nuclease-free water (无核酸酶水) to 20 µL to 50 µL

5 X Phusion HF or GC Buffer 4 µL 10 µL 1X

10 mM dNTPs 0.4 µL 1 µL 200 µM

10 µM Forward Primer (正向引物) 1 µL 2.5 µL 0.5 µM

10 µM Reverse Primer (反向引物) 1 µL 2.5 µL 0.5 µM

Component (组分) 20 µL Reaction 50 µL Reaction Final Concentratio

Template DNA (模板 DNA) Variable (see below) Variable (see below) < 250 ng

DMSO (optional) (可选) (0.6 µL) (1.5 µL) 3%

Phusion DNA Polymerase 0.2 µL 0.5 µL 1.0 units/50 µL PCR

To determine the amount of template DNA:

确定模板 DNA 的量：

DNA Amount (量)

Genomic (基因组) 50 ng–250 ng

plasmid or viral (质粒或病毒) 1 pg–10 ng

Thermocycling conditions for a routine PCR:

常规 PCR 的热循环条件：

Step (步骤) T [°C] Time (时间)

Initial Denaturation (初始变性) 98°C 30 seconds

25-35 Cycles (循环)

Denaturation (变性) 98°C 5-10 seconds

Annealing (退火) T annealing of the primers (45-72°C) 10-30 seconds

Elongation (延伸) 72°C Depending on the length of t

Final extension (最终延伸) 72°C 5-10 minutes

Hold (保温) 4-10°C  

Trouble shooting (故障排除):

Gradient PCR: make a gradient in the PCR machine to test different annealing temperatures.

Sometimes primers work better at a lower or higher temperature as the theoretical best temperature.

梯度 PCR: 在 PCR 仪中设置梯度以测试不同的退火温度。有时引物在比理论最佳温度更低或更高的温度下工作效果更好。

Quick-step PCR: try omitting the annealing step in the PCR program if the annealing temperature of the primers is too high (>68 °C). Just run your sample by cycling between the denaturing (98°C) and extension (72°C) step.

两步法 PCR (Quick-step): 如果引物的退火温度太高 (>68 °C)，尝试省略 PCR 程序中的退火步骤。只需在变性 (98°C) 和延伸 (72°C) 步骤之间循环运行样品。

Touch-down PCR: increasing specificity of PCR reactions (less nonspecific amplification) by starting with a higher annealing temperature and gradually reducing it through e.g. 10 cycles until it reaches the calculated annealing temperature of the primers or some degrees below. Follow the standard

Thermocycling conditions (Touch-down example):

热循环条件（降落 PCR 示例）：

Step T [°C] Time

Initial Denaturation 98°C 30 seconds

10 Cycles Auto delta: -0.5°C

Denaturation 98°C 5-10 seconds

Annealing T annealing + 2°C 10-30 seconds

Elongation 72°C Depending on length / 15-30s/kb

25 Cycles

Denaturation 98°C 5-10 seconds

Annealing T annealing – 3°C 10-30 seconds

Elongation 72°C Depending on length / 15-30s/kb

Final extension 72°C 5-10 minutes

Hold 4-10°C

PCR clean-upPCR 产物纯化

GeneJET PCR Purification Kit

GeneJET PCR 纯化试剂盒

Add a 1:1 volume of Binding Buffer to completed PCR mixture and mix thoroughly by pipetting up and down. (Color should be yellow. If the color of the solution is orange or violet, add 10 μL of 3 M sodium acetate, pH 5.2 solution and mix.)

向完成的 PCR 混合物中加入 1:1 体积的结合缓冲液（Binding Buffer），并通过上下吹打充分混合。

（颜色应为黄色。如果溶液颜色为橙色或紫色，加入 10 μL 3 M 乙酸钠 pH 5.2 溶液并混合。）

💡 注释: 结合缓冲液中含有 pH 指示剂。DNA 结合硅胶膜需要酸性高盐环境，黄色表示 pH 正确。

• Optional: if the DNA fragment is ≤ 500 bp, add a 1:2 volume of 100 % isopropanol (e.g., 100 μL of isopropanol should be added to 100 μL of PCR mixture combined with 100 μL of Binding

Buffer). Mix thoroughly.

• 可选：如果 DNA 片段 ≤ 500 bp，加入 1:2 体积的 100% 异丙醇（例如，向 100 μL PCR 混合物与 100 μL 结合缓冲液的混合液中加入 100 μL 异丙醇）。充分混合。

Note. If PCR mixture contains primer-dimers, purification without isopropanol is recommended.

• Repeat this step, until the entire solution has been added to the column.
• 重复此步骤，直到所有溶液都已加入柱中。

Add 50 μL of Elution Buffer or Milli-Q to the center of the GeneJET purification column membrane and centrifuge for 1 min.

向 GeneJET 纯化柱膜的中心加入 50 μL 洗脱缓冲液或 Milli-Q 水，并离心 1 分钟。

• Note. For low DNA amounts the elution volumes can be reduced to a volume between 20-50 μL.
• 注意：对于低 DNA 量，洗脱体积可以减少到 20-50 μL 之间。
• If DNA fragment is >10 kb, prewarm Elution Buffer to 65 °C before applying to column.
• 如果 DNA 片段 >10 kb，在加到柱上之前将洗脱缓冲液预热至 65 °C。
• You can resuspend the flow-through containing the plasmid on the same spin column membrane and repeat steps the last two steps to get a slightly higher plasmid yield.
• 你可以将含有质粒的流穿液重新加到同一个离心柱膜上，并重复最后两个步骤，以获得稍高的质粒产量。

Isopropanol method

异丙醇法 Extraction buffer:

提取缓冲液：

200 mM Tris-HCl pH 7.5 250 mM NaCl 25 mM EDTA 0.5 % SDS

Protocol:

实验方案：

Resuspend in ± 20 µL MilliQ 用约 20 µL MilliQ 水重悬。

Gel extraction with a kit

试剂盒胶回收 E.Z.N.A Gel Extraction Kit (Omega Bio-tek)

E.Z.N.A 凝胶提取试剂盒 (Omega Bio-tek)

Before starting:

开始之前：

Protocol:

• Monitor the pH of Gel/Binding Buffer mixture by color (if it turns orange/red, add 5 µM sodium acetate pH 5.2).
• 通过颜色监测凝胶/结合缓冲液混合物的 pH 值（如果变为橙色/红色，加入 5 µM 乙酸钠 pH 5.2）。

• Check if SPW Buffer is diluted with 100 % ethanol prior use.
• 使用前检查 SPW 缓冲液是否已用 100% 乙醇稀释。

• To increase the DNA yield, preheat the Elution buffer or Mili-Q water to 65 °C.
• 为增加 DNA 产量，将洗脱缓冲液或 Mili-Q 水预热至 65 °C。

• To increase the DNA yield: repeat the last 3 steps by re-suspending the same DNA elution fraction onto the same column membrane.
• 为增加 DNA 产量：通过将相同的 DNA 洗脱液重新加到同一柱膜上来重复最后 3 个步骤。

Low melting agarose gel extraction

低熔点琼脂糖凝胶回收 (原文此标题下无内容，通常参考常规胶回收步骤或说明该步骤在特定实验中与常规方法类似)

Agarose gel electrophoresis

琼脂糖凝胶电泳 1 x TAE running buffer recipe can be diluted from a 50 X TAE stock solution with pH around 8.5:

1 x TAE 电泳缓冲液配方可以由 pH 约 8.5 的 50 X TAE 储备液稀释而成：

50 X TAE stock For 1 L:

50 X TAE 储备液 (1 L):

Tris-buffer (121.14 g/mol): 242 g (Final: 2 M) 0.5 M EDTA stock solution (pH 8): 100 mL (Final: 50 mM)

Acetic acid 100 %: 57.1 mL (Final: 1 M)

DNA gel electrophoresis:

DNA 凝胶电泳：

▪ For several general approaches in our lab, a 1 % gel is appropriate ▪ 对于我们实验室的几种常规方法，1% 凝胶是合适的 ▪ For small DNA fragments (< 750 bp), try using a higher % (e.g. 2 %) ▪ 对于小 DNA 片段 (< 750 bp)，尝试使用较高百分比（例如 2%） ▪ For separating large DNA fragments (> 5.000 bp), try using a lower % (e.g. 0.8 %) ▪ 对于分离大 DNA 片段 (> 5,000 bp)，尝试使用较低百分比（例如 0.8%）

▪ Use around 30 mL for small gels ▪ 小胶约用 30 mL ▪ Use around 60 mL for medium gels ▪ 中胶约用 60 mL ▪ Use around 80 mL for large gels (two layers of combs) ▪ 大胶（双层梳子）约用 80 mL

Pour the mixture into a prepared gel holder including the gel comb and add a droplet (1-2 µL) of

Load your samples.

上样。

Load a DNA ladder (e.g. 10 Kb-ladder: 4.5 µL, at one or both sides of your sample).

上载 DNA Ladder（例如 10 Kb-ladder：4.5 µL，在样品的一侧或两侧）。

RNA gel electrophoresis:

RNA 凝胶电泳：

Note: RNA is easily degraded by RNases which are omnipresent in the gel tank or the buffers. Also use filter tips to prevent aerosols being blown in your samples. Make sure all products are clean by rinsing them with RNase-AWAY. Wear gloves at all times.

注意：RNA 很容易被凝胶槽或缓冲液中无处不在的 RNases（RNA 酶）降解。还要使用滤芯吸头以防止气溶胶吹入样品中。确保所有物品都通过 RNase-AWAY 清洗干净。全程佩戴手套。

💡 注释: DEPC 水处理可灭活水中的 RNase。

Add the normal DNA ladder.

加入常规 DNA Ladder。

Restriction enzyme digestion

限制性内切酶消化（酶切） A full list of restriction enzymes available in the PLT lab can be found in “Documents - BIOSYST - Van de Poel Lab/PPL-Lists/Restricton enzymes PPL.xlsx”

PLT 实验室中可用的限制性内切酶的完整列表可以在“Documents - BIOSYST - Van de Poel Lab/PPL-

Lists/Restricton enzymes PPL.xlsx”中找到。

A typical restriction digest using ThermoFisherScientific restriction enzymes:

使用 ThermoFisherScientific 限制性内切酶的典型酶切反应：

Component Amount

dH2O Up to 20 µL

10X Buffer 2 µL (1X)

DNA Up to 1 µg

Restriction Enzyme (10 units) 1 µL each

💡 注释: 酶储存在甘油中，过多的甘油（>10%）会抑制酶活性或导致“星号活性”（Star activity，即非特异性切割）。

Scientific - BE 的限制性内切酶反应条件）。

• Longer incubation times are needed when buffer compatibility is not optimal.
• 当缓冲液兼容性不是最佳时，需要更长的孵育时间。

Ligation

连接

T4 ligation

T4 连接 Set up the following reaction in a pcr-tube on ice

在冰上的 PCR 管中设置以下反应

Component 20 μL Reaction

T4 DNA Ligase Buffer (10X)* 2 μL

Vector DNA (50 ng) Use NEBioCalculator to calculate molar ratios

Insert DNA Use NEBioCalculator to calculate molar ratios

Nuclease-free water to 20 μL

T4 DNA Ligase 1 μL

💡 注释: 缓冲液中含有 ATP，为连接反应提供能量。ATP 遇冷易沉淀，需充分混匀，否则连接效率极低。

Incubate in a PCR machine:

在 PCR 仪中孵育：

• For cohesive (sticky) ends, incubate at 16 °C overnight or room temperature for 10 minutes.
• 对于粘性末端，在 16 °C 过夜孵育或在室温下孵育 10 分钟。

💡 注释: 16°C 是连接酶活性和氢键稳定性之间的平衡点。室温下酶活性高但氢键不稳定，低温下氢键稳定但酶活性低。

• For blunt ends or single base overhangs, incubate at 16 °C overnight or room temperature for 2 hours. (high concentration T4 DNA Ligase can be used in a 10 minute ligation).
• 对于平末端或单碱基突出端，在 16 °C 过夜孵育或在室温下孵育 2 小时。（高浓度 T4 DNA 连接酶可用于 10 分钟连接）。
• Alternatively incubate at room temperature for 2 hours, followed by incubation at 16°C overnight.
• 或者在室温下孵育 2 小时，然后在 16°C 过夜孵育。

Restriction-based cloning

基于酶切的克隆 This is a classical method by combining the unique cut-sites of two different restriction enzymes to clone in a DNA fragment in the vector of choice. The RE cut-sites for a PCR product are typically added to the primers (extra base pairs after the template specific primer part).

这是一种经典方法，通过结合两种不同限制性内切酶的独特切割位点，将 DNA 片段克隆到选定的载体中。PCR 产物的酶切位点通常添加到引物中（位于模板特异性引物部分之后的额外碱基对）。

Note: make sure these restriction sites are unique to the sites you need, avoid internal cut sites in both the vector and insert. In the case the RE can cut in your template, either use other RE or perform site directed mutagenesis.

注意：确保这些限制性位点是你所需的唯一位点，避免载体和插入片段内部出现切割位点。如果酶在模板内部有切点，请使用其他酶或进行定点突变。

• The restricted insert can be isolated by using a PCR purification kit (see PCR clean-up).
• 酶切后的插入片段可以使用 PCR 纯化试剂盒分离（见 PCR 纯化）。
• The restricted vector backbone will have to be gel extracted, therefore it is recommended to use 1 µg of DNA template for its digestion.
• 酶切后的载体骨架必须进行胶回收，因此建议使用 1 µg DNA 模板进行消化。

Note: If you are going to use only one restriction enzyme for both ends, or enzymes that have compatible overhangs or no overhangs after digestion, you will need to use a phosphatase to prevent re-circularization of the backbone plasmid. Therefore, treat the vector with a phosphatase after RE digestion and prior to the ligation step (or prior to the gel purification step). CIP (calf alkaline phosphatase) or SAP (shrimp alkaline phosphatase) are commonly used phosphatases.

注意：如果你打算在两端只使用一种限制性内切酶，或者使用消化后具有兼容突出端或无突出端的酶，你需要使用磷酸酶来防止骨架质粒的自环化。因此，在酶切消化后和连接步骤之前（或胶纯化步骤之前），用磷酸酶处理载体。CIP（小牛碱性磷酸酶）或 SAP（虾碱性磷酸酶）是常用的磷酸酶。

💡 注释: 连接酶需要 5' 磷酸基团才能工作。磷酸酶去除载体末端的 5' 磷酸，使其无法自我连接（自连）， 只能与带有 5' 磷酸的插入片段（未处理过）连接。

Gateway cloning

Gateway 克隆 Official protocol for multiple Gateway cloning:

多重 Gateway 克隆的官方方案：

https://www.thermofisher.com/be/en/home/life-science/cloning/gateway-cloning.html

The protocol is described for cloning a promotor and gene of interest using two entry vectors:

Primer design for cloning of promoter in pDONRP4P1:

在 pDONRP4P1 中克隆启动子的引物设计：

Primer Sequence

attB4-Promoter-F GGGGACAACTTTGTATAGAAAAGTTGxxxxxxxxxxxxxxxxxxxx

attB1r-Promoter-R GGGGACTGCTTTTTTGTACAAACTTGxxxxxxxxxxxxxxxxxxxx

Note: in some cases, there are regulatory elements in the first intron of the gene, and in this case, you include the first intron in the sequence of the promotor. Make sure the start site is non-functional, so that the actual start site is in the fusion protein.

注意：在某些情况下，基因的第一个内含子中存在调控元件，在这种情况下，你要将第一个内含子包含在启动子序列中。确保该起始位点无功能，以便实际起始位点位于融合蛋白中。

Primer design for cloning of coding sequences in pDONR221 or pDONR207:

在 pDONR221 或 pDONR207 中克隆编码序列的引物设计：

For the gene-specific sequences:

对于基因特异性序列：

For the reverse primer, it depends if you want to add a C-terminal tag (e.g. fluorescent marker like

GFP).

对于反向引物，这取决于你是否要添加 C 端标签（例如荧光标记，如 GFP）。

Primer Sequence

attB1-CDS-F GGGGACAAGTTTGTACAAAAAAGCAGGCTTCatgxxxxxxxxxxxx

attB2-CDS-R GGGGACCACTTTGTACAAGAAAGCTGGGTCxxxxxxxxxxxxxx

Before starting:

开始之前：

BP reaction (Gateway™ BP Clonase™ II Enzyme mix):

BP 反应 (Gateway™ BP Clonase™ II Enzyme mix):

(功能：将 PCR 产物重组入入门载体 pDONR)

Put 150 ng entry vector (e.g. pDONR221 or pDONR227 or pDONRP4P1) in a PCR tube.

将 150 ng 入门载体（例如 pDONR221 或 pDONR227 或 pDONRP4P1）放入 PCR 管中。

Add attB-PCR product (40-100 fmol).

加入 attB-PCR 产物 (40-100 fmol)。

Optional: Add 0.5 µL of Proteinase K solution to terminate the reaction; incubate for 10 min at 37°C.

For calculating the fmol PCR-product needed for the BP reaction, use the following formula:

计算 BP 反应所需的 fmol PCR 产物，使用以下公式：

f mol × N × 660 fg 1 ng ng = × 6 fmol 10 fg ​ ​

N: size of DNA in bp (DNA 的大小，单位 bp)

For further details see: (Link in original) 更多详情请见：（原文中有链接）

LR reaction (Gateway™ LR Clonase™ II Enzyme mix):

LR 反应 (Gateway™ LR Clonase™ II Enzyme mix):

(功能：将入门克隆中的片段重组入目的载体 pDEST)

Optional: Add 0.5 µL of Proteinase K solution to terminate the reaction; incubate for 10 min at 37°C.

Note: For multiple Gateway cloning:

注意：对于多重 Gateway 克隆：

GreenGate cloning

GreenGate 克隆 GreenGate cloning is based on BsaI mediated golden gate cloning, but adapted with a suite of plasmids for use in plants. The strategy consists of first making 6 entry plasmids (A – F modules, corresponding to promoter, N-terminal tag, CDS, C-terminal tag, terminator and a plant resistance gene) using BsaI cloning.

GreenGate 克隆基于 BsaI 介导的 Golden Gate 克隆，但通过一套质粒进行了调整以用于植物。该策略包括首先使用 BsaI 克隆制作 6 个入门质粒（A – F 模块，分别对应启动子、N 端标签、CDS、C 端标签、终止子和植物抗性基因）。

Then, these 6 plasmids and the destination plasmid are taken to a single tube Greengate reaction, where they are digested with BsaI and ligated with T4 DNA ligase. The standard GreenGate protocol results in a single expression casette, but it is also possible to have multiple expression casettes.

然后，将这 6 个质粒和目的质粒放入单管 Greengate 反应中，在那里它们被 BsaI 消化并用 T4 DNA 连接酶连接。标准的 GreenGate 方案产生单个表达盒，但也可以拥有多个表达盒。

The benefits of Greengate are that it's modular, scar-free and easy to make single trascriptional units.

We have the original GreenGate kit (Addgene kit 1000000036; https://www.addgene.org/kits/lohmanngreengate/; Lampropoulos A, Sutikovic Z, Wenzl C, Maegele I, Lohmann JU, et al. (2013) GreenGate - A

Novel, Versatile, and Efficient Cloning System for Plant Transgenesis. PLOS ONE 8(12): e83043.

Addgene 或共享的 –80 excel 表中获得。我们还在 B 模块中添加了 GFP。

GreenGate vector design and layout. The GreenGate system six modules represent the plant promoter (1), an N-terminal tag (2), the coding sequence of the gene of interest (3), a C-terminal tag (4), the plant terminator (5) and the plant resistance cassette (6).

GreenGate 载体设计和布局。 GreenGate 系统的六个模块代表植物启动子 (1)、N 端标签 (2)、感兴趣基因的编码序列 (3)、C 端标签 (4)、植物终止子 (5) 和植物抗性盒 (6)。

GreenGate entry vector (入门载体)

The multiple cloning site of pUC19 has been replaced by two BsaI recognition sites (magenta scissors), the respective overhangs for each module type, and a counter-selectable ccdB gene.

💡 注释: ccdB 基因编码一种毒性蛋白。如果在克隆过程中未被插入片段替换，含有该基因的载体将杀死大肠杆菌。这保证了长出的菌落都是成功插入了片段的阳性克隆。

GreenGate destination vector (目的载体)

The GreenGate cloning system uses a pGreen-IIS based destination vector.

GreenGate 克隆系统使用基于 pGreen-IIS 的目的载体。

The empty destination vector consists of a counter-selectable ccdB-cassette between the LB and

Making entry modules:

制作入门模块：

PCR is used to amplify the region of interest and add appropriate BsaI sites for cloning. The following sequences are added in front of the gene specific region of the F primer, and in front of the reverse complement of the gene specific region for the R primer.

PCR 用于扩增感兴趣的区域并添加适当的 BsaI 位点进行克隆。以下序列被添加到 F 引物的基因特异性区域之前， 以及 R 引物的基因特异性区域的反向互补序列之前。

F: 5' AACAGGTCTCANNNN (nn) - Gene specific region -3'

R: 5'-AACAGGTCTCANNNN – Gene specific region reverse complement 3'

GGTCTC is the BsaI recognition site.

GGTCTC 是 BsaI 识别位点。

AACA was added because the enzyme does not cut if the restriction site is at the extreme ends of

Greengate reaction:

Greengate 反应：

Set up the following in PCR tubes:

在 PCR 管中设置以下内容：

1.5 µl each entry vector 100 ng/µl (每个入门载体 1.5 µl) 1 µl destination plasmid 100 ng/µl (目的质粒 1 µl) 1.5 µl cutsmart buffer 1.5 µl ATP 10 mM 1 µl T4 DNA ligase 1 µl BsaI-HF

To 15 µl with H2O

💡 注释: 37°C 是 BsaI 酶切的最佳温度，16°C 是 T4 连接酶的最佳温度。循环进行可以不断切割-连接。

Golden Gate 的特点是产物不再包含酶切位点，因此循环反应会使反应向产物方向进行。

Multiple transcriptional units多转录单元

Two transcriptional units两个转录单元

For two transcriptional units, two intermediate vectors, pGGM000 and pGGN000, and two adapter molecules are used.

对于两个转录单元，使用两个中间载体 pGGM000 和 pGGN000 以及两个接头分子。

The two supermodules are then combined with the destination vector in a second step in a standard

More than two transcriptional units超过两个转录单元

Therefore, in each subsequent GreenGate round either the uncleavable oligo duplex can be used to prepare the plasmid for the insertion of another expression cassette or the plasmid can be finalized by adding a plant resistance cassette to the reaction.

因此，在随后的每一轮 GreenGate 中，可以使用不可切割的寡核苷酸双链体来准备质粒以插入另一个表达盒，或

者可以通过向反应中添加植物抗性盒来完成质粒。

The FHadapter used along with the oligo duplex separates the individual expression units in the final construct and thus can be freely designed to act as a spacer of arbitrary length and sequence minimizing the risk of mutual interference of the expression behavior.

与寡核苷酸双链体一起使用的 FHadapter 分隔最终构建体中的各个表达单元，因此可以自由设计为任意长度和序列的间隔区，从而最大限度地降低表达行为相互干扰的风险。

Gibson cloning

Gibson 克隆

Gibson primer designGibson 引物设计

Protocol Samuel Miller Lab

Samuel Miller 实验室方案

Gibson assembly primers are broken down in two parts: primer sequence and overlap sequence. The primer sequence should be designed using traditional characteristics in mind (i.e. Tm° values, G/C ratio, and G/C anchors). The overlap sequence needs to have between 20 – 150 bp homology to insert or vector. A good recommended start is a primer of 60 bp in total length: 30 bp of vector and 30 bp of insert.

Gibson 组装引物分为两部分：引物序列和重叠序列。引物序列的设计应考虑传统特征（如 Tm 值、G/C 比和 G/C 锚点）。重叠序列需要与插入片段或载体具有 20 – 150 bp 的同源性。建议从总长度为 60 bp 的引物开始：30 bp 的载体序列和 30 bp 的插入序列。

A: 5’ – (30 bp vector/ 30 bp insert) – 3’ and D: 5’ – (30 bp insert/ 30 bp vector) – 3’

C: 3’ – (30 bp vector/ 30 bp insert) – 5’ and B: 3’ – (30 bp insert/ 30 bp vector) – 5’

💡 注释: PCR 产物是非甲基化的，而细菌提取的质粒（模板）是甲基化的。DpnI 酶特异性降解甲基化

DNA，从而消除背景质粒。

Gibson reaction

Gibson 反应

Reaction Stock Preparation反应储备液制备

Prepare 5x ISO buffer: 6 mL of this buffer can be prepared by combining the following:

制备 5x ISO 缓冲液：通过混合以下成分可制备 6 mL 此缓冲液：

3 mL of 1 M Tris-HCl pH 7.5 150 μL of 2 M MgCl2 60 μL of 100 mM dGTP 60 μL of 100 mM dATP 60 μL of 100 mM dTTP 60 μL of 100 mM dCTP 300 μL of 1 M DTT 1.5 g PEG-8000

💡 注释: PEG-8000 是分子拥挤剂，模拟细胞内环境，增加分子碰撞概率，提高酶促反应效率。

300 μL of 100 mM NAD

💡 注释: NAD 是 Taq 连接酶的辅因子。

Add water to 6 ml

加水至 6 ml

Prepare master mixture. Combine the following:

制备母液。混合以下成分：

320 μL 5x ISO buffer 0.64 μL of 10 U/μL T5 exonuclease

💡 注释: T5 外切酶负责从 5' 端回切 DNA，产生 3' 单链突出端用于退火。

20 μL of 2 U/μL Phusion polymerase

💡 注释: Phusion 聚合酶负责填补退火后的空隙。

160 μL of 40 U/μL Taq ligase

💡 注释: Taq 连接酶负责连接缺口（Nick），形成完整的环状 DNA。

Add water to 1.2 ml 加水至 1.2 ml

Reaction Procedure反应步骤

Thaw a 15 μL assembly mixture aliquot and keep on ice until ready to be used.

解冻一份 15 μL 组装混合物，并在使用前保持在冰上。

Gibson limitations and obstaclesGibson 的局限性和障碍

A major limitation (especially in high GC-rich templates such as in P. aeruginosa) to Gibson assembly is that the termini of the DNA sequence fragments to be assembled, should not have stable single stranded DNA secondary structure, such as a hairpin or a stem loop, or repeated sequences, as this would directly compete with the required single-stranded annealing/priming of neighboring assembly fragments.

Gibson 组装的一个主要限制（特别是在高 GC 含量的模板中，如铜绿假单胞菌）是，待组装的 DNA 序列片段的末端不应具有稳定的单链 DNA 二级结构，如发夹或茎环，或重复序列，因为这将直接与相邻组装片段所需的单链退火/引发竞争。

Site directed mutagenesis (SDM)

定点突变 (SDM) Blunt end ligation-based SDM methods:

基于平末端连接的 SDM 方法：

SDM using blunt end ligation results in a blunt end PCR product, which is circularized by ligation. First, primers are designed to incorporate the desired mutation (substitution, deletion or insertion). The plasmid to mutagenize is used as a template for PCR, and the resulting blunt end PCR product is ligated.

使用平末端连接的 SDM 会产生平末端 PCR 产物，该产物通过连接环化。首先，设计引物以引入所需的突变（替换、缺失或插入）。将待诱变的质粒用作 PCR 模板，并将得到的平末端 PCR 产物进行连接。

Because the PCR product is missing the 5' phosphate, this needs to be added using a kinase. To avoid returning the original vector used as a template in the PCR, the sample should be digested using DpnI, which only cleaves methylated DNA and not the PCR product. Finally, the blunt end PCR product should be circularized using a ligase.

因为 PCR 产物缺少 5' 磷酸基团，所以需要使用激酶添加磷酸基团。为了避免 PCR 中用作模板的原始载体复原， 样品应用 DpnI 消化，它只切割甲基化的 DNA 而不切割 PCR 产物。最后，平末端 PCR 产物应使用连接酶环化。

Independent enzymes SDM (独立酶法 SDM)

• Perform PCR using a high-fidelity polymerase (Q5 or Phusion).
• 使用高保真聚合酶（Q5 或 Phusion）进行 PCR。
• PCR clean PCR product.
• 纯化 PCR 产物。
• Set up DpnI (Fast digest Thermo FD1703) digestion on PCR product:
• 对 PCR 产物设置 DpnI (Fast digest Thermo FD1703) 消化：

Component Amount

DNA Up to 1 µg

10X FastDigest buffer 2 µL

DpnI 1 µL

Nuclease free H2O To 20 µL

• Incubate 37 °C for 5 mins, inactivate at 80 C for 5 mins in a PCR machine.
• 在 PCR 仪中 37 °C 孵育 5 分钟，80 °C 灭活 5 分钟。
• Clean digested product with a PCR clean up column.
• 用 PCR 纯化柱纯化消化后的产物。
• Set up the T4 polynucleotide kinase (NEB M0201) reaction:
• 设置 T4 多核苷酸激酶 (NEB M0201) 反应：

Component Amount

DNA Up to 300 pmol of 5’ termini*

T4 PNK Reaction Buffer 10X 5 µL

ATP 10 mM 5 µL

T4 PNK 1 µL 10 units

Nuclease free H2O To 50 µL

Use NEBioCalculator for calculating mass of DNA to add

使用 NEBioCalculator 计算需添加的 DNA 质量

moles of dsDNA ends = mass of dsDNA (g)/((length of dsDNA (bp) x 615.96 g/mol/bp) + 36.04

g/mol) x 2

The T4 PNK buffer can be substituted for T4 DNA ligase buffer. The 1X T4 DNA ligase buffer contains 1 mM ATP, so ATP does not need to be added. In this case, the product does not need to be cleaned before ligation, but supplemental ATP should be added to the kinase reaction to a final concentration of 1 mM.

T4 PNK 缓冲液可以用 T4 DNA 连接酶缓冲液代替。1X T4 DNA 连接酶缓冲液含有 1 mM ATP，因此不需要额外添加 ATP。在这种情况下，产物在连接前不需要纯化，但应向激酶反应中补充 ATP 至终浓度为 1 mM。

• Incubate at 37 °C for 30 minutes in PCR machine.
• 在 PCR 仪中 37 °C 孵育 30 分钟。
• Inactivate the reaction at 65 °C for 20 minutes.
• 在 65 °C 下灭活反应 20 分钟。
• Clean product with a PCR clean up column.
• 用 PCR 纯化柱纯化产物。
• Set up T4 ligase (NEB M0202) ligation:
• 设置 T4 连接酶 (NEB M0202) 连接：

Component Amount

DNA 1 µL of 50 ng

T4 Ligase Buffer 10X 2 µL

T4 Ligase 1 µL

Nuclease free H2O To 20 µL

• Incubate at 16 °C overnight or at room temperature for 10 minutes.
• 在 16 °C 过夜孵育或在室温下孵育 10 分钟。

• Heat inactivate at 65°C for 10 mins.
• 在 65°C 下热灭活 10 分钟。
• Transform 1-5 µL into appropriate E. coli strain.
• 将 1-5 µL 转化到适当的大肠杆菌菌株中。

• Primer should be designed with 5’ ends annealing back-to-back. It is recommended to use the

NEB online design software (NEBaseChanger).

• 引物设计应使 5' 端背对背退火。建议使用 NEB 在线设计软件 (NEBaseChanger)。
• Perform a PCR using a high-fidelity polymerase (Q5 or Phusion).
• 使用高保真聚合酶（Q5 或 Phusion）进行 PCR。

PCR MasterMix:

Component 25µL RXN 12.5µL RXN Final conc.

Q5 Hot Start High-Fidelity 2X MasterMix 12.5µL 6.25µL 1X

10 µM Forward Primer 1.25µL 0.625µL 0.5 µM

10 µM Reverse Primer 1.25µL 0.625µL 0.5 µM

Template DNA (1-25 ng/µL) 1 µL 0.5µL 1-25 ng

Nulcease-free water 9µL 4.5µL

Cycling conditions:

Step Temperature Time

Initial Denaturation 98 °C 30 sec

25 Cycles

(Denaturation) 98 °C 10 sec

(Annealing) 50-72 °C* 10-30 sec

(Extension) 72 °C 20-30 sec/kb

Final Extension 72 °C 2 min

Hold 4-10 °C

• Do a PCR clean-up using the kit.
• 使用试剂盒进行 PCR 纯化。
• Set up the following KLD (Kinase, Ligase, DpnI) reaction on ice:
• 在冰上设置以下 KLD（激酶、连接酶、DpnI）反应：

Component Amount

PCR product 1 µL

2X KLD reaction buffer 5 µL

10X KLD enzyme mix 1 µL

Nuclease free H2O 3 µL

• Mix well by pipetting up and down and incubate at room temperature for 5 minutes.
• 通过上下吹打充分混合并在室温下孵育 5 分钟。
• Transform 5 µL of KLD reaction to 50 µL appropriate competent cells.
• 将 5 µL KLD 反应液转化到 50 µL 适当的感受态细胞中。

Gibson-based SDM methods:

基于 Gibson 的 SDM 方法：

Gibson based SDM also requires designing primers incorporating the desired mutation, but the resulting PCR product should have complementary overlapping ends of 15-30 bp. Then an exonuclease chews back the 5' end, complementary regions anneal, DNA polymerase fills in the gaps within each annealed fragment, and ligase seals the nicks. The benefit of this approach is that it can also be used to assemble multiple fragments in one go.

基于 Gibson 的 SDM 也需要设计包含所需突变的引物，但得到的 PCR 产物应具有 15-30 bp 的互补重叠末端。

然后外切酶回切 5' 端，互补区域退火，DNA 聚合酶填充每个退火片段内的空隙，连接酶封闭缺口。这种方法的优点是它也可以用于一次组装多个片段。

NEB HIFI (E2621)

• Perform PCR using a high-fidelity polymerase (Q5 or Phusion)
• 使用高保真聚合酶（Q5 或 Phusion）进行 PCR
• PCR clean the PCR product
• 纯化 PCR 产物
• Set up the following reaction on ice:
• 在冰上设置以下反应：

Recommended Amount of Fragments Used for Assembly

Component 2–3 Fragment Assembly* 4–6 Fragment Assembly**

Recommended DNA Molar Ratio vector:insert = 1:2 vector:insert = 1:1

Total Amount of Fragments 0.03–0.2 pmols* (X μL) 0.2–0.5 pmols** (X μL)

NEBuilder HiFi DNA Assembly Master Mix 10 μL 10 μL

Nuclease free H2O 10-X μL 10-X μL

Total Volume 20 μL †† 20 μL ††  

Optimized cloning efficiency is 50–100 ng of vector with 2-fold excess of each insert. Use 5-fold molar excess of any insert(s) less than 200 bp. Total volume of unpurified PCR fragments in the assembly reaction should not exceed 20 %. To achieve optimal assembly efficiency, design 15- 20 bp overlap regions between each fragment.

优化的克隆效率是用 50–100 ng 载体和 2 倍过量的每个插入片段。对于小于 200 bp 的任何插入片段，使用 5 倍摩尔过量。组装反应中未纯化 PCR 片段的总量不应超过 20%。为获得最佳组装效率，在每个片段之间设计 15-20 bp 的重叠区域。

† Control reagents are provided for 5 experiments.

† 提供的对照试剂可供 5 次实验使用。

• Incubate samples in a thermocycler at 50 °C for 15 minutes (when 2 or 3 fragments are being assembled) or 60 minutes (when 4–6 fragments are being assembled).
• 在热循环仪中 50 °C 孵育样品 15 分钟（组装 2 或 3 个片段时）或 60 分钟（组装 4-6 个片段时）。
• Store samples on ice or at –20 °C for subsequent transformation.
• 将样品储存在冰上或 –20 °C 以备后续转化。
• Transform 2-5 uL of assembled product to appropriate cell strain.
• 将 2-5 uL 组装产物转化到适当的细胞菌株中。

Restriction enzyme-based SDM methods:

基于限制性内切酶的 SDM 方法：

Restriction enzyme based SDM makes use of type II restriction enzymes (we have BsaI, AarI and BbsI).

Primers can be designed to introduce the appropriate mutation with internal restriction sites. This can be designed so that the recognition sites are cut out, leaving complementary sticky ends which are ligated. The benefit of this method is that multiple products can be ligated together, but it is best if there are no internal sites for the restriction enzymes which are used.

可以设计引物以通过内部限制性位点引入适当的突变。设计时可确保识别位点被切除，留下互补的粘性末端进行连接。此方法的优点是可以将多个产物连接在一起，但最好是所用的限制性内切酶没有内部切点。

Type II restriction enzyme mutagenesisII 型限制性内切酶诱变

• Perform PCR using a high-fidelity polymerase (Q5 or Phusion). Set up the following reaction:
• 使用高保真聚合酶（Q5 或 Phusion）进行 PCR。设置以下反应：

Component 25µL RXN 12.5µL RXN Final conc.

Q5 Hot Start High-Fidelity 2X MasterMix 12.5µL 6.25µL 1X

10 µM Forward Primer 1.25µL 0.625µL 0.5 µM

10 µM Reverse Primer 1.25µL 0.625µL 0.5 µM

Template DNA (1-25 ng/µL) 1 µL 0.5µL 1-25 ng

Nuclease-free water 9µL 4.5µL

• Cycling conditions:
• 循环条件：

Step Temperature Time

Initial Denaturation 98 °C 30 sec

25 Cycles

(Denaturation) 98 °C 10 sec

(Annealing) 50-72 °C* 10-30 sec

(Extension) 72 °C 20-30 sec/kb

Final Extension 72 °C 2 min

Hold 4-10 °C

*For mutagenic primers, please use the Ta provided by the online NEB primer design software

• PCR clean the PCR product
• 纯化 PCR 产物
• Digest PCR product with appropriate type II restriction enzyme and DpnI (to digest template DNA that is methylated). A typical restriction digest using ThermoFisherScientific restriction enzymes:
• 用适当的 II 型限制性内切酶和 DpnI（消化甲基化的模板 DNA）消化 PCR 产物。使用

ThermoFisherScientific 限制性内切酶的典型酶切反应：

Component Amount

dH2O Up to 20 µL

10X Buffer* 2 µL (1X)

DNA Up to 1 µg

Restriction Enzyme (10 units) 1 µL each

• PCR clean PCR product
• 纯化 PCR 产物
• Ligate using T4 DNA ligase
• 使用 T4 DNA 连接酶进行连接

Component Amount

DNA 1 µL of 50 ng

Component Amount

T4 Ligase Buffer 10X 2 µL

T4 Ligase 1 µL

Nuclease free H2O To 20 µL

• Transform 1 µL of ligation to appropriate cells.
• 将 1 µL 连接产物转化到适当的细胞中。

Escherichia coli transformation大肠杆菌转化

Chemical transformation化学转化

• Do not vortex or pipet up and down!
• 不要涡旋或上下吹打！

💡 注释: 感受态细胞非常脆弱，剧烈震荡会破坏细胞。

• A lower volume of DNA will cause a lower dilution/contamination risk.
• 较小的 DNA 体积会降低稀释度/污染风险。

• If you use bought competent cells, reduce incubation time to 30 seconds.
• 如果你使用购买的感受态细胞，将孵育时间减少到 30 秒。

• Alternatively, use SOC medium or low salt LB to increase efficiency.
• 或者，使用 SOC 培养基或低盐 LB 以提高效率。

Incubate for 1 hour at 37 °C in a shaker.

在摇床中 37 °C 孵育 1 小时。

💡 注释: 此步骤用于浓缩细胞，以便在平板上接种更多菌量，适用于转化效率低的情况。

💡 注释: 卫星菌落（Satellite colonies）通常在长时间培养后出现在主菌落周围，它们不含质粒，是依靠主菌落降解抗生素生存的。

Competent cells available in the lab:

实验室现有的感受态细胞：

Depending on your plasmid of interest, choose between the following competent cells:

根据你感兴趣的质粒，在以下感受态细胞中进行选择：

• E. coli TOP10: F- mcrA (mrr-hsdRMS-mcrBC) 80lacZ M15 lacX74 recA1 ara 139 (ara-leu)7697

galU galK rpsL (StrR) endA1 nupG

▪ Similar to DH5a, streptomycin resistant, engineered for high transformation efficiency of

high-copy number plasmids

▪ 类似于 DH5a，对链霉素具有抗性，专为高拷贝数质粒的高转化效率而设计。

💡 注释: Top10 对 CcdB 毒蛋白敏感。如果转化含有 ccdB 的空载体（如未重组的 Gateway 入门载体），细菌会死亡。

• E. coli DB3.1: gyrA462 endA1 ∆(sr1-recA) mcrB mrr hsdS20 glnV44 (=supE44) ara14 galK2 lacY1 proA2 rpsL20 xyl5 leuB6 mtl1

▪ Streptomycin resistant

▪ 链霉素抗性。

▪ ccdB resistant, so empty vectors can be transformed

▪ 对 ccdB 具有抗性，因此可以转化空载体。

💡 注释: DB3.1 含有突变的 gyrA 基因，使其对 CcdB 毒蛋白免疫。扩增带有 ccdB 基因的空载体时必须使用此菌株。

• E. coli BL21: fhuA2 [lon] ompT gal [dcm] ΔhsdS

▪ Used for non-T7 expression

▪ 用于非 T7 表达。

▪ Suitable for transformation and protein expression

▪ 适用于转化和蛋白质表达。

💡 注释: BL21 缺乏蛋白酶（Lon 和 OmpT），适合表达外源蛋白，防止蛋白被降解。

Agrobacterium tumefaciens transformation

根癌农杆菌转化 Transformation of electrocompetent A. tumefaciens cells using electroporation:

使用电穿孔法转化农杆菌电击感受态细胞：

💡 注释: 电击转化对盐分非常敏感。盐分过高会导致电弧（Arcing，即短路爆杯），不仅转化失败，还可能损坏仪器。

Wipe ice/any residual water from the cuvettes before transferring it into the electroporator.

在将其转移到电穿孔仪之前，擦去电击杯上的冰/任何残留水。

💡 注释: 杯体外部的水会导致电流从外部通过，引发短路。

• Electroporation settings (电穿孔设置): (原文此处留白，通常农杆菌设置为 2.2 kV, 25 µF, 200-400

Ω)

💡 注释: 农杆菌生长较慢，且电击造成细胞壁损伤，需要较长的复苏时间（2-4小时），比大肠杆菌（1 小时）更长。

• Spin cell cultures down for 2 min at 3000 x g, remove supernatant and dissolve pellet in residual culture and plate the remaining culture on a separate selection plate.
• 将细胞培养物以 3000 x g 离心 2 分钟，去除上清液，用残留的培养物溶解沉淀，并将剩余的培养物涂布在单独的选择平板上。

Making competent cells

制备感受态细胞 Heat-shock competent Top 10 E. coli cells:

热激 Top 10 大肠杆菌感受态细胞：

Media (培养基):

250 mL 2x YT broth in 500 mL flask:

500 mL 烧瓶中的 250 mL 2x YT 肉汤：

4 g Bacto tryptone (4 g 细菌胰蛋白胨) 2.5 g Bacto yeast extract (2.5 g 细菌酵母提取物) 1.25 g NaCl (1.25 g 氯化钠)

Water up to 250 mL (加水至 250 mL)

PH to 7.0 with NaOH (用 NaOH 调节 pH 至 7.0)

Autoclave (高压灭菌)

50 mL 1x YT broth:

50 mL 1x YT 肉汤：

0.4 g Bacto tryptone 0.25 g Bacto yeast extract 0.125 g NaCl

Water up to 50 mL pH to 7.0 with NaOH

Autoclave

TS buffer (TSB; prepare fresh on the day, when the competent cells are being made):

TS 缓冲液 (TSB; 在制备感受态细胞的当天新鲜制备):

7.5 mL 2 x YT broth (final conc. 1X) 7.5 mL 2 x YT 肉汤（终浓度 1X） 129 µL 5 M NaCl (final conc. 43 mM) 129 µL 5 M NaCl（终浓度 43 mM） 150 µL 1 M MgCl2 (final conc. 10 mM) 150 µL 1 M MgCl2（终浓度 10 mM） 1.5 g polyethylene glycol (PEG) 4000 (final conc. 10 % (v/v)) 1.5 g 聚乙二醇 (PEG) 4000（终浓度 10 % (v/v)） 750 µL DMSO (final conc. 5 % (v/v)) 750 µL DMSO（终浓度 5 % (v/v)） 3 mL glycerol (final conc. 20 % (v/v)) 3 mL 甘油（终浓度 20 % (v/v)）

Water up to 15 mL 加水至 15 mL Sterile filter the buffer and keep cool until needed

除菌过滤缓冲液并保持冷却直到需要使用

For all steps, work in a laminar flow to keep the cells sterile 对于所有步骤，在层流罩中操作以保持细胞无菌

Day 1:

Day 2:

Day 3:

From here on, keep the cultures/cells always on ice 从这里开始，始终将培养物/细胞保持在冰上

💡 注释: OD600 0.3-0.6 处于对数生长期（Log phase），此时细菌状态最活跃，细胞壁通透性最好， 转化效率最高。

Resuspend the cells in 10 mL cold TSB (2.5 mL per falcon tube).

用 10 mL 冷 TSB（每个 Falcon 管 2.5 mL）重悬细胞。

Electrocompetent GV3101 Mp90 (pSoup-P19) Agrobacterium tumefaciens cells:

GV3101 Mp90 (pSoup-P19) 根癌农杆菌电击感受态细胞：

GV3101 Mp90 (pSoup-P19) from ZellBio. (Link in original) 来自 ZellBio 的 GV3101 Mp90 (pSoup-P19)。（原文有链接）

Day 1:

Inoculate A. tumefaciens colony (derived from a freshly streaked LB plate containing the appropriate antibiotics) in 3 mL LB culture with antibiotics at 28°C and 200 rpm (5 µg/mL rifampicin for A.

Day 2:

Day 3:

Vortex each falcon with 10 mL ice cold dH20, mix it and top up to 50 mL.

（原文可能有误，意为充分洗涤）用 10 mL 冰冷 dH20 涡旋每个 Falcon 管，混合并加满至 50 mL。

💡 注释: 这一系列繁琐的“离心-重悬”是水洗步骤，目的是彻底去除培养基中的盐分，防止电击时短路。

Colony PCR

菌落 PCR Colony PCRs can be used as an initial test to confirm success of the cloning approach before inoculating the transformants in liquid culture. For this, select a region for not more than 1 kb that you want to amplify. The primers that you use for amplifying the region of interest should be insert- and vectorspecific (e.g. forward primer binds within the insert, reverse primer binds in the vector backbone).

菌落 PCR 可用作初步测试，在将转化子接种到液体培养物之前确认克隆方法是否成功。为此，选择一个不超过 1 kb 的区域进行扩增。你用于扩增感兴趣区域的引物应为插入片段和载体特异性的（例如，正向引物结合在插入片段内，反向引物结合在载体骨架中）。

💡 注释: 使用一条载体引物和一条插入引物可以验证插入片段的方向是否正确，并避免空载体的假阳性。

Depending on the number of colonies you want to test, prepare a master mix with the ThermoScientific

DreamTaq Green PCR Master Mix (2X).

根据你要测试的菌落数量，使用 ThermoScientific DreamTaq Green PCR Master Mix (2X) 准备母液。

MasterMix for colony PCR:

菌落 PCR 母液：

Component Amount Final conc.

DreamTaq Green PCR Master Mix (2X)* 6.25 µL

Forward primer (10 µM stock conc.) 0.625 µL 0.5 µM final conc.

Reverse primer (10 µM stock conc.) 0.625 µL 0.5 µM final conc.

MilliQ water Up to 12.5 µL

*no need to use a high fidelity polymerase (TAQ is sufficient)

*不需要使用高保真聚合酶（TAQ 就足够了）

Adding material of the bacterial colonies into the PCR vials containing the PCR mixture:

将细菌菌落材料加入含有 PCR 混合物的 PCR 管中：

💡 注释: 菌量过多会抑制 PCR 反应。

PCR settings:

PCR 设置：

Perform the PCR according to the manufacturer's instructions, to the length of the fragment you want to amplify and the annealing temperature of the primers.

根据制造商的说明、你要扩增的片段长度和引物的退火温度进行 PCR。

💡 注释: 高温初始变性除了激活热启动酶外，主要功能是裂解细菌释放质粒。农杆菌细胞壁更厚，需要更长时间。

After performing the PCR reaction in a thermo cycler, run the PCR in on a 1 % agarose gel together with a 1 kB DNA ladder. Afterwards, visualize the DNA bands with UV light. If the DNA bands are in accordance with the expected fragment size, you can inoculate the bacterial colony into LB medium with the respective antibiotic for further downstream processing (e.g. plasmid prep).

在热循环仪中进行 PCR 反应后，将 PCR 产物与 1 kB DNA Ladder 一起在 1% 琼脂糖凝胶上进行电泳。之后，用紫外光观察 DNA 条带。如果 DNA 条带与预期的片段大小一致，你可以将细菌菌落接种到含有相应抗生素的 LB 培养基中，进行进一步的下游处理（例如质粒制备）。

Sequencing (Sanger)

Step 1: Prepare your samples in 1.5 mL reaction tubes 第 1 步：在 1.5 mL 反应管中准备样品

Template requirements:

模板要求：

Template Concentration

Plasmids 100 ng/µL

PCR products

200 - 500 bp 10 ng/µL

500 - 1,000 bp 20 ng/µL

1,000 - 2,000 bp 40 ng/µL

FlexiRun: 40 µL of template DNA

FlexiRun: 40 µL 模板 DNA Ready2Run: Pre-pipetted primer (10 µL DNA + 5 µL primer [5 µM])

Ready2Run: 预加引物（10 µL DNA + 5 µL 引物 [5 µM]）

Step 2: Place your order at LGC 第 2 步：在 LGC 下单 (Link in original)

At “sequencing ordering”, you will find below “single sample” the option for “Ready2 / Flexi

Run”.

在“测序订购”处，你会在“单一样品”下方找到“Ready2 / Flexi Run”选项。

• In case you chose Flexi Run, indicate which primer should be used by LGC.
• 如果你选择 Flexi Run，请注明 LGC 应使用哪种引物。

Step 3: Analyze your sequences with Geneious 第 3 步：用 Geneious 分析你的序列

Book working station in advance via clustermarket.

通过 clustermarket 提前预订工作站。

Antibiotics

Filter-sterilize in the laminar flow and aliquot into 1.5 mL tubes.

在层流罩中进行除菌过滤，并分装到 1.5 mL 管中。

💡 注释: 培养基太热（>55°C）会使抗生素失效。

Antibiotic (抗生素) Stock concentration (储备浓度) Working concentration (工作

Ampicillin (氨苄青霉素) 100 mg/mL 100 µg/mL

Carbenicillin (羧苄青霉素) 100 mg/mL 100 µg/mL

(alternative to ampicillin)

Chloramphenicol (氯霉素) 25 mg/mL (dissolved in EtOH) 25 µg/mL

Kanamycin (卡那霉素) 50 mg/mL 50 µg/mL

Spectinomycin (大观霉素) 50 mg/mL 50 µg/mL

Gentamycin (庆大霉素) 50 mg/mL 30 µg/mL

Tetracycline (四环素) 10 mg/mL 10 µg/mL

Rifampicin (利福平) 50 mg/mL (dissolved in DMSO or MeOH) 5-20 µg/mL

Cefotaxime (头孢噻肟) 100 mg/mL 100 µg/mL  

（完 / End of Manual）