Cloning and Mutagenesis System

Cloning FAQs


Q: How to choose the right cloning vector?

A: For Taq-amplified PCR products, choose T cloning vector. For Pfu-amplified PCR products, choose Blunt cloning vectors, or T cloning vector after adding a single deoxyadenosine (A) (the result will be compromised compared with ligating Blunt vectors directly). For plasmid templates, dual antibiotic resistance vectors are recommended.


Q: What is the appropriate amount of inserted fragments?

A:

· The amount of fragment addition can be adjusted according to the length of the fragment. The optimal vector-to-insert molar ratio is 1:7.

· It can be roughly calculated at the ratio of "1 kb- to-20 ng" (e. g. 20ng for 1 kb inserted DNA, ; 30 ng for 1.5 kb inserted DNA) (The amount can be quantified roughly by TransGen's DNA molecular weight standard on the gel.)

· The maximum amount of fragment addition is 4 μl, and do not exceed this limit even if the fragment concentration is very low. The minimum amount of fragment addition is 0.5 μl, which can be replenished with sterile water for easy operation.

· Reaction temperatures range: 20°C to 37°C . The optimal reaction temperature is 25°C.

 

Q: How long should the reaction last?

A:

· Reactions for most PCR products within 1 kb can be completed in 5 minutes under room temperature.

· There are several situations in which reaction time can be extended for better results:

1. Inserts with special structures (high AT content/ high GC content/ inverted repeat sequence): 10-20 minutes.

2. Inserts from gel extraction or A overhangs addition: 10-15 minutes (this condition applies to fragments up to 3 kb).

3. PCR products larger than 3 kb: 15-20 minutes.

 

Q: How long can the ligated mixture be stored?

A: The ligated mixture can be stored in a refrigerator of -20°C or 2-8°C, and should be used within one week.

 

Q: Unable to cleave the restriction sites in the multiple cloning site.

A:The screened colonies come from the plasmid templates rather than the ligation mixture. It is recommended to use double antibiotic-resistant vectors and choose the resistance that plasmid templates don’t contain for screening.

 

Q: Few colonies or low cloning efficiency?

A:

There are many factors that affect the number of colonies and cloning efficiency, such as the transformation efficiency of competent cells, the quality of the inserts, the structure of the gene, the length of the inserts, insert-to-vector molar ratio, etc. If you find that the number of colonies is low or the cloning is inefficient, you can try the following methods:

· The transformation efficiency of competent cells has significant influence on the number of colonies, and their relationship is not linear. For example, when the transformation efficiency of the competent cells is 1 x 109 cfu /µg DNA, the number of colonies is 1000. But when the transformation efficiency is 1 x 108 cfu / µg DNA, the number of colonies is less than 100. In order to ensure a high number of colonies, please choose competent cells Trans1-T1 with high transformation efficiency.

· Use fresh PCR products: PCR products should be stored at 2-8℃ and used within 1-2 days. For isolated DNA from gel extraction, the UV light exposure time should be minimized and the reaction time should be extended appropriately (10-15 minutes).

· When the concentration of the target fragment is very low, concentrate to ensure more chances of molecular collision during reaction.

· Toxic genes: Use Trans10 competent cells.

· Genes with special structures (high AT content/ high GC content/ inverted repeat sequence): extend reaction time to 10-20 minutes.


Q: Failure to verify the transformants by PCR?

A:

When expected amplified products, or vector self-ligation have not been observed by PCR with universal primers, this means PCR has failed. Optimize the PCR reaction conditions again or extract the plasmids which can be used as templates for amplification or digested by restriction enzyme to identify colonies containing transformants.

 

Q: Colonies show light blue or "Fish eye"

A: If the insert does not affect the LacZ reading frame, or the insert is too short, the colonies appear light blue or "Fish eye", and can still be verified as usual.


pEASY Cloning Vectors
pEASY® -T1 Cloning Kit
pEASY® -Blunt Cloning Kit
pEASY® -T1 Simple Cloning Kit
pEASY® -Blunt Simple Cloning Kit
pEASY® -T3 Cloning Kit
pEASY® -Blunt3 Cloning Kit
pEASY® -T5 Zero Cloning Kit
pEASY® -Blunt Zero Cloning Kit
pEASY® -Uni Seamless Cloning and Assembly Kit
pEASY®-Basic Seamless Cloning and Assembly Kit
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Mutagenesis Systems
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