Hi
I used PstI restriction enzyme to cut my PCR product with a RFLP site in it. I could'nt see any digested product. It may be because of the polymorphism or the enzyme could be inactive. I don't have plasmid to check the activity of my enzyme. What other way can I use to check whether my enzyme is active or not?
borrow some pcr product from a colleague which has a cutsite in it ( use NEBcutter at new england biolabs to check sequences and the band will become 2 or more bands. Or cut genomic dna and run the uncut and cut product on an 0.8% agarose gel and the high MW dna should become a smear of lower MW dna smeard over most of the length of the gel
borrow some pcr product from a colleague which has a cutsite in it ( use NEBcutter at new england biolabs to check sequences and the band will become 2 or more bands. Or cut genomic dna and run the uncut and cut product on an 0.8% agarose gel and the high MW dna should become a smear of lower MW dna smeard over most of the length of the gel
Regardless, for genotyping experiments (e.g. PCR-RFLP) you need to simultaneously run all 3 controls (wt/wt, mut/mut, mut/wt). Thus you need to sequence a few PCR products from gDNA of a number of individuals to obtain these DNAs. Sometimes the minor allele frequency is very low, and thus having the mut/mut DNA is not feasible.
what sizes are your fragments .are you running a non cut control to see if there is a size change.sometimes if there is only a small fragment it does not trap much ETBR and you do not see it and the larger fragment getting smaller is a better indicator
also make sure that you load enough pcr product so that you can see one quarter of it after digestion. PCR is not very efficient in the later cycles and if you are cutting a heterozygote then 1/4 will be normal/normal, 1/4 will be mutant/mutant and one half will be heteropuplexed normal mutant and of all of these options only one of the homozygous strands will cut with restriction enzymes as the bubble in the heteroduplex will not allow digestion
You may have a problem in RFLP optimization conditions. Try to use the follwing protcol we set in our lab.
for 20 micron reaction use: \
10 micron PCR products
1 micron enzyme
5 buffer
4 micron nuclease free water.
What size is your PCR product? You need to run the uncut and the cut product on the same gel to confirm whether the restriction enzyme worked or not. If the ezyme didnt cut the PCR product, there could be several reasons like something wrong with the enzyme, reaction condition, etc. As some other people suggested sequence your PCR and do the restriction site analysis to see if any polymorphism on the restriction site. Hope this help.
The easiest and the most available way is to treat any plant/animal genomic DNA with the enzyme and compare with the non-treated sample of the same DNA on agarose gel. good luck
You can use any vector in your lab or borrow from some other lab containing PstI cutting site. and also run uncut plasmid as control.
I agree with Namdar Moradi, the simplest way is just to take a good genomic DNA (gDNA) of any species and digest it with your enzyme (PstI), and run a gel. If your enzyme is active, the lane on the gel with treated gDNA sample should be 'smear', the lane with un-treated gDNA sample should be a single good band. This is also how people do the Southern analysis-digest the gDNA first.
Attached picture is an example for you.
@ Vijayashree Priyadharsini, So, did you find out whether your restriction enzyme works?
if you have NEBCutter program you can identify the possible sites for your enzyme.
you can also use BioEdit and GeneiousPro programs too
I accepted all recomendation. Just I remembered when I was working with the NdeI Ihad the same problem. It was because of salt concentration. Some restriction enzymes are sensitive to protein or salt concentration. So, If you have any DNA extraction procedure it is good to try with another kit. Also, I would like to ask about your internal control. Did you have it? What is happening to it?
Hi all,
Tried to digest genomic DNA with the enzyme..got a smear!
Thanks for your valuable time and support.
Hi @Vijayashree,
May I know whether you finally managed to see any band of your digested PCR product? SInce you know that your enzyme is still active, after treating your PCR product with the restriction enzyme, did you manage to see and bands after all? If you do, could you please tell me how you do it? Because, currently, I am having the same problem as well.
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