My plasmid (pJET1.2) contains an insert of around 2969bp. The total size of plasmid 5942 bp. I am cutting it with AhdI, SgrAI and BssHII. Desired bands are of 2969, 1632 and 1432bp. I want to clone 2968bp gene. But I was getting bands of 3000,1550, 1350bp and also very light band of 625bp. when I gel-purified 3000bp band and run on gel then it was showing an extra band of 2700bp. Somehow I was able to clone this 3000bp but colony PCR shows a band of 2700bp instead of 2969bp. Also, RE confirmation shows a band size of 2700bp instead of 2969bp. Please anyone help me to solve this problem as I have repeated the experiment with so many times but getting similar results. My insert contains secondary sites that are similar to the recognition site of SgrAI but not exactly similar.
hi
you can order your target gene in desired vector according to codon optimality( gene synthesis company ) .
Note1 : 2969bp not differ size with 2700bp on agarose gel .
Why cut with three enzymes?
good luck
thank you @Reza Hadavi,
if we use two enzymes (SgrA1 and BssHII) then two bands of equal size get released which are inseparable. We have limited time and resources so we are unable to reorder.
Any other solutions, please suggest.
As Reza said, the mobility difference between 2700 and 2969 may be hard to distinguish on an Agarose gel. It sounds like the primers that you are using for colony PCR are both on your vector; without spending much money, you could order a primer that binds to your insert and perform a colony PCR using a forward primer on your insert and a reverse primer on your vector-- This would at least answer if the 2700 band is your insert or not, which may assist your troubleshooting. If it is indeed the correct band and you don't have the resources to redesign this study, you could try to shorten your restriction enzyme digestion time or see if the manufacturer of your SgrAI and BssHII enzymes has a recommendation for buffer additives or alternative buffer formulations that reduce star activity.
Are the 2700 and 2969 bands of similar intensity in the gel?
I am wondering if the insert has a high GC or AT region that is forming a loop and making the same product run at different sizes due to being a different shape. Also are you sure that other sites are only similar to the Sgr recognition site as this site contains 2 redundant sites Y and R
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