I did a colony recently. PCR a 1.5kb target gene with a poly(a) tail, followed the pGEM-t vector ligation, and then transforming the competent cell, finally using PCR, enzyme test and sequencing using T7 promoter strategy. Quizzically, only 1/3 of the sequencing result about 300bp were target gene, why does the sequencing result not match with the PCR or enzyme digest test?
There are multiple reasons and you may feel frustrated. The most common reason is due to contamination. Cloning kits these days are extremely straight forward and efficient. Look at your kit is not expired and work under laminar flow and very clean aseptic technique, do not even talk when plating. Purify your insert and when running the PCR product to be cloned use autoclaved buffer. We use to recycle agarose and buffers for routine gels (we are a GREEN LAB) but when cloning we clean it all. Cheers!
From your question I am not clear whether you mean that a third of your recombinant clones have the correct insert and the other two thirds do not or that all clones have the same insert that has one third correct sequence contiguous with two thirds that is the wrong thing.
If the problem is the former, then this you mat be able to do better by optimising the PCR conditions. Try using a touchdown PCR approach. But if you only need one or a few clones then do you actually need a better ratio of correct clones to incorrect? It is useful to remember that "Best is often the enemy of good enough".
If the problem is an unexpected product with 300bp of correct sequence followed by something else - then maybe you have clones an exon linked to an intron from genomic DNA. I notice you say "the gene has a polyA tail". If there is a polyA tail, then this started as RNA- genes don't have polyA tails, which are a feature of mRNAs. So if you are working from mRNA did you make a cDNA intermediate. If you did not but are actually cloning a gene fragment from DNA, did you allow for introns?
thanke you, David Richard Sargan
Actually, i did pcr of a virus gene. In each cycle, the polymerase i used could added poly A tail which contributed to T vector ligation in the following steps. The vector has the EcoR I sites between the inserts.
To confirm the pcr products, i did a plasmid pcr and EcoR I digest. The results were in accord with expectation. The final sequence results showed that only a small ratio colones were correct. I blast these wrong results with the target or vector gene, it turned out to be short fragment. I am very confusing.
hi, Joey Ma
competent cell's activity did influence the result, i compared the new made cells with the long placed one. the latter did need more hours to recover.
Does it exist the possible that numbers of ligation products transformed into the same competent cell, thus causing the results that the short fragment had more advantage upon replicating.
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