Hi, I have two enzymes - EcoR1 and Kpn1. Both the enzyme expirations dates in 2010. Can I use these two enzymes for cloning? Will it work and, if yes, will there be proper restriction overhangs? Because my insert is just 700 base long and I find it very difficult to get it cloned. After double digestion, vector gives desired single band. but I doubt if the overhangs are maintained.The PCR product is good. my competent cells and transformation protocol is fine. and i use new vial of T4 DNA ligase.
Thanks in advance ...
I would have tried, if it works it works. You will see on a gel if it worked, as a band of the size you want.
You can try them first but I used lately KpnI expired too and it worked perfectly. I just put the double of the recommended amount in the protocol.
you can add a small trail .....mostly if it in right temp it wont fail.
Not sure what you are doing here. Are you cutting a vector with Kpn1/R1 and then the pcr product with the same enzymes to clone into it? If so check both enzymes can linearise the vector on their own. Also are you doing a vector only control ligation? The critical thing is that all the vector is cut by both enzymes or it will self ligate. If I had doubts about an enzyme working 100% I would use CIP on the vector even for a double digest.
It depends. Better if you done a small experiment whether it provides you desired result or not. All the best
I used many enzymes with last two years expiry. This is worked out for me. I strongly recommended you go for small experiment before doing a large scale experiment.
I have used enzymes up to 5 years after expiration, PROVIDED they were stored properly and not subjected to repeat freeze/thaw cycles. I agree with the researcher who said that you should do a small scale experiment first to make sure that the enzymes work.
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