i need your expert opinion.
if we are cloning a gene which is amplified by a cDNA that is having some traces of gDNA. Following TA cloning and sub cloning into a destination vector or protein expression vector,
1) will there be any effect on constitutive expression of gene?
2) or on protein product?
provided that post-transcriptional modification may occur in vivo?
Hi Qari,
I assume that you isolate your amplicon after PCR from a gel slice and than perform the TA cloning. If your bandsize correlates with the predicted size and you don't see any other band than gDNA contamination had no effect. You could sequence your amplicon to make sure it is the right one. Once you cloned the right amplicon into the vector there won't be any effect of gDNA on protein product.
Best regards
I agree with Soner. During cloning of DNA-fragment into a vector, an additional sequence of gDNA can be seen during the selection of clones (after restriction analysis or PCR analysis, the cloned fragment becomes heavier), and sequence-level correctness is usually checked by sequencing. It is very important to make a preliminary computer analysis of restriction sites, the position of the initiator and stop codon in the fragment of interest.
I agree with miss oxana. As sequencing is best way to check the correctness of our desired gene sequence. so just make sure that you have done all the steps carefully and have chosen the right band size from gel slice.
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