cloning of PCR product
Amita Misra
Monday, 15 December 2008 18:41 UTC
Hi, I am trying to ligate PCR product in an expression vector. I’ve restriction enzyme sites in my PCR product. I use to cut the vector as well as the PCR product with restriction enzyme and then doing the gel elution. But after ligating the vector and insert I am not able to get any colony following transformation.
Plz suggest me what to do.
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Replies
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Anonymous
You should check that you still have DNA after gel purification, and make sure you are ligating the right ratios of insert to vector together. Check that in the ligation buffer you can still see the ATP (white fluffy bits) as freeze/thawing can reduce the activity of the buffer. Check that you are growing the bugs on the right selection plates and do a positive control to check that your competent cells are actually competent.
Hope this helps, I’m also stuck in cloning limbo at the moment!
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thanx for ur suggestion but I tried all this. I always check DNA after gel purification and it looks good to me. I tried insert vector ratio from 3:1 to 5:1 and even more and vice versa. Ligation buffer is also ok as it gives positive result with controls. I m using all controls to satify any mistake. I think problem starts after gel purification.Are u using electroporation or using chemical competent cells.which one will be better?
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Anonymous
Hello, there are several things may go wrong. You should have good competent cells with good efficiency. Transformation condition shall be good. It means early these cells and conditions have worked for you. Type of transformation will not matter much (in my hand), but in electro-competent cells amount of salt in ligation mix matters (some times might need ethanol precipitation). These are less important as u need only couple of positive colonies. Load the ligation mix on a good gel to see the ligated product. If you dont see then may be you need to use new ligation buffer or old one supplemented with ATP and also DTT if needed. Some times if you have used very less DNA you might not see at all. You need better digestion of vector and PCR product. Your or your colleagues previous experiments with same enzymes should tell you that enzymes and buffers are good/bad. We can not produce controls many time as told in big protocol books. Mix the DNA depending on how you see them on gel like 1:3 of vector:insert. Take vector just enough to see on a fresh Etbr gel (dont go for quantification). If you think gel-purification is troubling you, if your PCR products are single bands and excising very few base pairs from vectors go for column purification, and follow same protocol. I have told my experience it might be different from books. Hope it helps. Good luck
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Hi Amita, you first check whether your ligation has worked out fine.. for this you can put the ligation mix PCR using vector specific (Like T7 or M13) forward/gene specific reverse OR vice versa.. So that u can check the expected amplicon size… Ok All the best
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