产品内容

产品简介

快速连接试剂盒在室温（25℃）反应5分钟可完成DNA粘性末端或平齐末端的连接反应。试剂盒含有Quick T4 DNA Ligase和为快速高效DNA连接优化的2×Quick Ligation Reaction Buffer。快速连接的连接效率相当于用T4 DNA Ligase 进行常规连接1小时。快速连接产物可直接用于常规的细菌转化实验。

注意事项

1．本试剂盒能使大多数连接反应在25℃条件下5分钟甚至更短时间内达到反应终点， 增加反应时间反应效率不会增强。如用快速连接反应1小时后，转化效率会明显降 低；如25℃快速连接反应过夜，则转化效率会下降到75%。 

2．2×Quick Ligation Reaction Buffer含有ATP，使用前置于冰上使其融化后充分混匀， 建议初次使用分装成小管冻存，避免其反复冻融影响DNA连接效率。 

3．由于T4 DNA Ligase中含有甘油，比较粘稠容易挂壁，建议使用之前短暂离心将液 体收集到管底，取样时枪头尽量不要深入液面太深，以免粘在枪头上造成损失。 

4．如快速连接产物用于电转，快速连接反应体系中的PEG会影响电转效率，建议使用离心柱将连接产物进行DNA纯化后再进行电转。

使用方法

1．按以下体系配制反应液：

*Insert DNA的使用量：Vector DNA和 Insert DNA的摩尔比一般为1:3-1:8，可根据实验情况选择适当的Vector DNA和 Insert DNA的摩尔数比。DNA摩尔数计算方式： DNA摩尔数（nmol）=DNA质量（ng）/ (660 daltons×插入DNA碱基数bp)。

2．轻轻混合，短暂离心。25℃反应5分钟。 

注意：反应时间不要超过15分钟，否则会降低连接效率。 

3．勿进行加热失活反应。瞬间离心，将管壁上的溶液收集到管底。 

注意：因缓冲液中含有PEG，加热失活会显著降低转化效率。 

4．反应结束后，将DNA连接产物存放于0-4℃，而后进行转化实验；也可将DNA连接 产物置于-20℃保存。 

注意：用化学法转化时，连接产物的加入量不要超过感受态细胞体积的10%。 

5．将连接产物热击转化50 μl感受态细胞或取1-2 μl连接产物电击转化50 μl感受态细胞。 

注意：1）用化学法转化时，连接产物的加入量不要超过感受态细胞体积的10%。 

2）如快速连接产物用于电转，因快速连接反应体系中的PEG会影响电转效率，建议使用离心柱将 连接产物进行DNA纯化后再进行电转。

Product content

1. The reaction solution was prepared according to the following system:

*The amount of Insert DNA used: the molar ratio of Vector DNA and Insert DNA is generally 1:3-1:8, and the appropriate molar ratio of Vector DNA and Insert DNA can be selected according to the experimental situation.Calculation of DNA molar number: DNA molar number (nmol)=DNA mass (ng)/( 660daltons x number of inserted DNA bases bp).

2. mix gently and centrifuge briefly. react at 25°C for 5 minutes.

Note: The reaction time should not exceed 15 minutes, otherwise the connection efficiency will be reduced.

3. Do not perform heat inactivation reactions. Centrifuge instantly and collect the solution from the wall to the bottom of the tube.

Note: Heat inactivation significantly reduces transformation efficiency due to the presence of PEG in the buffer.

4. After the reaction, store the DNA ligation product at 0-4℃, and then carry out transformation experiments; you can also store the DNA ligation product at -20℃.

Note: When transforming by chemical method, do not add more than 10% of the volume of the receptor cell for the ligation product.

5. Heat shock the ligation product to transform 50 μl of receptor cells or take 1-2 μl of ligation product to electroshock transform 50 μl of receptor cells.

Note: 1) When transforming by chemical method, do not add more than 10% of the volume of the receptor cell for the ligation product.

(2) If the quick ligation product is used for electrotransformation, it is recommended to use a centrifugal column to purify the ligation product from DNA before electrotransformation because the PEG in the quick ligation reaction system will affect the efficiency of electrotransformation.