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 ...
Just try it first to make sure that the enzyme still work and cut the DNA you desired to cut. If it stored in recommended conditions, i think it still good to use.
Yes you should try to ligate it with variation of molar ratios of insert and vector.
After double digestion of 2 enzymes your vector was linearized. So it will visualized in agarose as single band
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.
Shouldn't be a problem... You can setup a single/double test digest as per your requirement, in desired buffer and you are all set to go if the digests work as per the expectation. Good Luck... :)
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