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restriction digestion of PCR product


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#1 maxmix

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Posted 12 January 2010 - 02:36 AM

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

#2 almost a doctor

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Posted 12 January 2010 - 02:57 AM

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

#3 phage434

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Posted 12 January 2010 - 07:32 AM

Have you purified your pcr product prior to digestion?

#4 microgirl

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Posted 12 January 2010 - 09:01 AM

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.

#5 maxmix

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Posted 12 January 2010 - 09:15 PM

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

#6 maxmix

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Posted 12 January 2010 - 09:16 PM

Have you purified your pcr product prior to digestion?

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

#7 maxmix

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Posted 12 January 2010 - 09:20 PM

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???

#8 phage434

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Posted 13 January 2010 - 05:45 AM

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?




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