I have recently started working on gene cloning I want to digest my PCR product and my plasmid with BamH1 and Nde1 but I am not able to digest them using 1 unit of enzyme for 10 micro gram of DNA. How to slove this issue.
As a general guide: one unit of enzyme will cut 1 µg of DNA in a 50 µL reaction in 1 hour.
So you may need to alter your protocol. In addition, the NEBcloner provides a service to optimise your enzyme activity: http://nebcloner.neb.com/#!/redigest
Hope this helps!
Mistakes in molecular biology are usually by being greedy. But when you learn most of the methods, you trust your protocols to give you the amount you want without being greedy. - So if you're going to clone a fragment into a vector, assuming that there are major difficulties you just need 1.0 ug (one microgram of plasmid, undigested). - Next, it's to choose the enzymes, here you have BamH1 and Nde1. I don't know which brand you're using but in NEB you use NEBuffer 3.1 to digest both targets without star activity. Next, it's to calculate volumes: for a 50uL reaction, I'd use 1.0 ug of indigested vector, 1 uL of BamH1 and 1 uL of Nde1, 5 uL of NEBuffer 3.1. So here you have a reasonable volume 50 uL, 1x buffer for a double digest, 1 ul of each enzyme (10 units) it's plenty. So a large volume allows to proper mixing and water loss, avoid excesses of DNA or its byproducts. The most important thing here is that all plasmid to be digested and thus if you use too much, some of it would be undigested and would give you high background of colonies. Next is to phosphatase the reaction, a good molecular biology grade shrimp AlkPhosp would do the job. You can add it directly to the digestion. When the time comes, inactivate all enzymes. Next, isolate the plasmid in a gel. Important to get rid of contaminants.
Which company are your enzymes from ? NEB Roche ? Fermentas ? Which buffer did you use ? Did you put both enzyme at the same time ? They are at least half a dozen of reasons why it didn't work
If it is restriction enzymes I would like to say the STAR activity and maybe the steps taken. (Enzymes having different activities than the original one) this could led to suboptimal conditions. Make sure those enzymes are set in the right place. Make sure you do not cut any valuable gene. WhatI don't know if you could use a marker (antibiotic) or maybe beta galactosidase, which turns the organism blue.
Well just one more question is popping up to my brain as I read other comments. How close to the end of your pCR product are your restriction site located ? You need at least an overhang of 5 nucleotides in order to get a proper cut and even so you still need very optimal conditions in order to be successful
In addition to all the comments above, how do you actually know that BamHI and NdeI are not cutting? If they are near the ends of the product you might never see any size shift.
I have problems with restriction digests in the past. It turned out that I was using the wrong buffer! Make sure your buffer is compatible with your enzymes.
Also what is the size of your plasmid and how long are you incubating your digests? you may need need a longer incubation time.
- Badges
- Science method