I am having trouble with cloning. My PCR product is 1kb. After ligation (with pGEM easy vector) into the plasmid, I transformed into JM109 competent cell. I can get white colonies but when I did the colony PCR it was false positive, just one colony was ok. Ratio is 3:1 (Insert: vector) and I left the ligation reaction over night at 4C. I tried with different accessions but the same DNA target and the result was the same in all samples.
Please what is wrong?.
I can think of three things that might go wrong.
1. Is your restriction digestion working out properly. Have you confirmed it by running on a gel.
2. Try out ligation at room temperature. At 4C i am afraid that enzyme will not be efficient. I always did ligation at RT itself worked perfectly for me.
3. What is the antibiotic that you are using. Small pipetting errors can lead to extensive variations. I have already faced difficulties because of this.
Please let me know if something works out thanks.
there can be many reasons but let me choose some of them:
-perform a control in your experiments to see whether the problem is relating to your ligation or transformation.
if it s because of transformation so check bacteria competency, transformation method, growth conditions
but if it s because of ligation so calculate your vector:insert molar ratio precisely. it is VERY important...putting too much DNA in your ligation reaction reduces the ligation efficiency so try not to exceed 200ng DNA in your ligation reaction.... ligation buffer is important since containing ATP, instead of doing several freeze and thaw, aliquot it to avoid ATP breakdown...do not forget that ligase needs free A at both ends of amplification products to ligate it to linear vector so important to choose the appropriate polymerase...
The antibiotic is Ampicillim. When I tried with a small DNA target (like less than 500 bp) but same sample and with the same conditions the transformation cell is successful.
I will tried with different ligation temperature and molar ratio.
Thank you.
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