Dear colleagues,
I am a Ph.D. student and I´m trying to build up an immune library derived from PBLs in a Fab format.
I have obtained cDNA from RNA. The first round of PCRs was performed to amplify the coding sequences and, a second-round for adding “bigger tails” in order to promote restriction.
I am trying to clone the amplicons into pComb3X vector, with a two steps cloning protocol (light and heavy chains). I have performed small-scale ligations and then, E.coli XL-1 Blue transformations. It worked for heavy chains but not for light ones and I don´t know what is going wrong.
I have tried: 1) different conditions for restriction (time and enzyme concentration) obtaining proper and clean digested DNAs; 2) different ligations (insert:vector ratio and DNA quantity); 3) different DNA volumes for transformations. The results have been disappointing, or no colonies after plating transformation or the grown colonies, once sequenced, present only the pComb3X with no insert.
The weird issue is that heavy chains are cloning correctly but not light ones.
Can anyone help me please? Thank you so much!
I'm not quite sure what is the difficulty with your process? Once you have the amplicon from your second PCR you can digest it with the same R.E as your vector(Phagemid).However, the caution point is you would potentially require good concentration of insert ( ~ 3ug) to produce 10^7 transformants and have a decent diverse library. Peopel mention the fact that electroporation efficiency needs to be > 75% to proceed to panning stage..Also try to clean up after every step be it gel or PCR purification.
https://res.mdpi.com/d_attachment/mps/mps-02-00017/article_deploy/mps-02-00017.pdf
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