I have cloned a 6.46 kb having restriction sites for enzymes BamHI and Not I in pcDNA3.1 vector. After colony pcr screening, the result shows positive result with primer specific to vector and insert region. However no insert size was detected by digestion. Note. ligation reaction was transformed into DH5a competent cells. I am not sure whether DH5a is suitable for large insert size. Thank you so much for kind suggestion.
6.4kb is not really that large, there should be no problem getting the clone and DH5a will be suitable (unless your insert has lots of repeat sequences).
It may be that you just have a mixture, the PCR detects the fraction with the insert but when you picked a colony you got one without an insert. I would just screen more plasmids.
Dear Michael J. Benedik,
I truly appreciate your suggestion. I screened approximately 120 colonies, however, I found 14 positive colonies and I double-digested them with BamHI and Not I. The result also showed no insert. 100 colonies are enough? or screening more? Thank you so much.
Do the digests of those 14 colonies look any different (maybe larger for example) than the others or the control? It might be a problem with the digestion for example.
I would suggest you take a few of the 14 colonies and restreak them for single colonies and test again.
Another trick you could try is to digest the ligation mix (or pool the DNA from the 14 colonies) and digest with one of the restriction enzymes that cut between the BamHI and NotI site, if those are unique and not found in your insert. That reduces the background of parent plasmids and selects for DNA that you want.
Thank you for your tips! They are very useful! i will try it again.
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