7 replies to this topic

[maxmix]

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.

[almost a doctor]

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.

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

[phage434]

Have you purified your pcr product prior to digestion?

[microgirl]

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.

[maxmix]

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.

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???

[phage434]

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?