I'd like to sequence a 600bp PCR product.
Here's my method:
1) PCR clean-up by QIAquick kit
2) ligation with T4 ligase
3) transformation with heat shock
4) incubate on LB plate overnight
There's just few colonies on plates, which step can I start with troubleshooting ?
Thank you for your advice.
I use TA cloning and I had 2 colonies in one plate.
Here're two additional questions:
(1) how to know the efficiency of competent cell
If I need to check the efficiency of competent cell, should I put the vector without insert DNA into competent cell, then incubate overnight and count colonies?
(2) how to check ligation efficeincy
As your advice, different ratios of vector and insert would affect the ligation result. I'd like to check it by changing the ratio. Can I measure vector's concentrations by nano drop?
To estimate transformation efficiency you may use any vector (pUC, pTZ, Bluescript etc), but you need to dilute it 1000-10000 fold (0.1 ng/ul concentration looks most convenient). Efficiency is OK if 0.1 ng of a vector gives at least 100 colonies. You may inspect ligation by electrophoresis, but I never do this. Absence of colonies in case of good cells indicates problems with ligation.
Nano drop properly works only in case of very pure and good quality DNA. I always estimate DNA concentration by comparison of band intensities on ETBR gel. TA cloning requires equal molar concentrations (you should take in account fragment size) of vector and insert.
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