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restriction digestion of PCR product - (Jan/12/2010 )

can one explane to me how to do restriction enzyme for PCR product by MnlI (biolab). I used: PCR product 10ul, 10x buffer:2ul, MnlI:1ul, water:17ul,,at 37C for 3hr but was bad???
another question I had faint DNA after agarose gel how to improve,, I used 150ng DNA. the primer was 20pmol,Mgcl:2.5Mm.
thank you all. :P

-maxmix-

maxmix on Jan 12 2010, 10:36 AM said:

can one explane to me how to do restriction enzyme for PCR product by MnlI (biolab). I used: PCR product 10ul, 10x buffer:2ul, MnlI:1ul, water:17ul,,at 37C for 3hr but was bad???
another question I had faint DNA after agarose gel how to improve,, I used 150ng DNA. the primer was 20pmol,Mgcl:2.5Mm.
thank you all. :P



Hi, for your restricition digest you are not adding enough 10x Buffer as your final volume is 30ul and you are only adding 2ul of buffer.

for your second question; I'm going to assume you are talking PCR, we need more details to help you.

cheers

-almost a doctor-

Have you purified your pcr product prior to digestion?

-phage434-

Clean your PCR product and quantify it before you digest. Then check out the recommendations on the directions of your enzyme - you need an excess of enzyme over DNA but too much enzyme is bad too. As someone pointed out you also didn't add enough buffer - or you added to much water. Adding BSA (which probably came with your enzyme) also can help your digest.

-microgirl-

microgirl on Jan 13 2010, 12:01 AM said:

Clean your PCR product and quantify it before you digest. Then check out the recommendations on the directions of your enzyme - you need an excess of enzyme over DNA but too much enzyme is bad too. As someone pointed out you also didn't add enough buffer - or you added to much water. Adding BSA (which probably came with your enzyme) also can help your digest.

hummm that is why,,i will try to add BSA.thank u so much

-maxmix-

phage434 on Jan 12 2010, 10:32 PM said:

Have you purified your pcr product prior to digestion?

yes I did. but maybe the buffer is not enough..thank u.

-maxmix-

almost a doctor on Jan 12 2010, 05:57 PM said:

maxmix on Jan 12 2010, 10:36 AM said:

can one explane to me how to do restriction enzyme for PCR product by MnlI (biolab). I used: PCR product 10ul, 10x buffer:2ul, MnlI:1ul, water:17ul,,at 37C for 3hr but was bad???
another question I had faint DNA after agarose gel how to improve,, I used 150ng DNA. the primer was 20pmol,Mgcl:2.5Mm.
thank you all. :D



Hi, for your restricition digest you are not adding enough 10x Buffer as your final volume is 30ul and you are only adding 2ul of buffer.

for your second question; I'm going to assume you are talking PCR, we need more details to help you.

cheers

oh, I will try to add more buffer, because I followed the Fermentase,sdn,phd. company protocol. 2nd Q: I am amplifying gene from human blood sample, the product was faint after agarose gel, is it because of the primer quantity, 20pmol???

-maxmix-

1) Do you still have DNA in your sample after pcr cleanup? Can you run a lane next to your digestion?
2) What do you mean when you say it "was bad"? It failed to cut? It was invisible? Cut at the wrong place?
Attached File

-phage434-