4 replies to this topic

[lotus]

Hi,
I tried to clone a 3.8 kb PCR product into teh pGEMTeasy vector. It was a blunt ended PCR product and so after purification, i added A tail to it and did ligation. I plated on LB amp plates with Xgal and IPTFG and got mostly blue colonies. Only a very few white ones. i screened the white ones by colony PCR using M13 Fwd and rev primers. I didn't get any band, except in 1, which had multiple bands. I grew up taht colony and did restriction digestion with NotI to release the insert. I should get 3.8 kb insert + 3 kb vector with NotI digestion. But i got 2.5 kb band + 6 kb band. so I think thsi colony is some non-specific DNA.

Anyway, how come white colonies don't have my insert? How can I increase amount of positive white colonies?

[Qundo12]

lotus, on Dec 12 2009, 04:36 AM, said:

Hi,
I tried to clone a 3.8 kb PCR product into teh pGEMTeasy vector. It was a blunt ended PCR product and so after purification, i added A tail to it and did ligation. I plated on LB amp plates with Xgal and IPTFG and got mostly blue colonies. Only a very few white ones. i screened the white ones by colony PCR using M13 Fwd and rev primers. I didn't get any band, except in 1, which had multiple bands. I grew up taht colony and did restriction digestion with NotI to release the insert. I should get 3.8 kb insert + 3 kb vector with NotI digestion. But i got 2.5 kb band + 6 kb band. so I think thsi colony is some non-specific DNA.

Anyway, how come white colonies don't have my insert? How can I increase amount of positive white colonies?

How did you purify your DNA? 3.8Kb is not that long for TA Cloning. My assumption is your purification was not good and the DNA fragmentation happened before the adding of the A tail. These short fragments easily ligated to your pGEM-T, generating the functional lacZ-a, so you've got blue colonies. Normally, the self ligation of pGEM-T is very low, so this is not the case. Since the short DNAs are very competitive, so they occupied most of the opened vectors. The plasmid you obtained maybe resulted from the complex ligation of 2 vector and a DNA fragment (maybe the reason why you had multiple bands).
Why didn't you use the DNA polymerase with 3'A overhang inserting activity? How did you add A tail to your products? The purification or tail adding step had problem, I think.
I suggest using the gel extraction for PCR product purification and clean up the reaction after adding A tail (what if the enzyme was still active in the presence of pGEM-T and add 3'A to one of the T overhang --> complementary to the opposite T --> self ligation + fill in). Ligate at 16 degree for 4h (normally I did that if the gene is larger than 3Kb) instead of RT for 1h as suggested by the protocol.

[lotus]

Thank you Quasimondo. I did not want to use Taq for the PCR because it cannot do proofreading. Do you think 3.8 kb can be amplified without errors using Taq?

[Vini]

lotus, on Dec 12 2009, 07:12 PM, said:

Thank you Quasimondo. I did not want to use Taq for the PCR because it cannot do proofreading. Do you think 3.8 kb can be amplified without errors using Taq?

Hi Lotus, its certainly not a good idea to use Taq pol for ur 3.8 kb amplification.....our experience with this polym for large amplicons has been really bad........we see many errors........it would be better then, that u use some other polym like , Phusion or Pfu....there are others also, which promise high efficiency n high fidelity long amplifications (Roche)

Best...

[Qundo12]

Sure, I didn't suggest you to use Taq because of that. In my lab, I have some other choices: LA Taq from Takara (for long PCR product, up to 25Kb) and HanTaq XL (since I'm in Korea, this is from a Korean company). They are high fidelity enough to clone a 3-8 Kb fragment (as my experience) and can add a A 3' tail. Normally I can get the correct sequence within 5 clones.