re-amplication of PCR product without internal primers

douglas l feinstein dlfeins at cumc.cornell.edu
Wed Aug 24 07:57:19 EST 1994


Subject: re-amplication of PCR product without internal primers
From: BHWFDUNG
Date: 18 Aug 1994 19:55:25 -0700
In article <01HG3ABNW3QQ8WXHAF at cphkvx.cphk.hk> , BHWFDUNG at CPHKVX.CPHK.HK
writes:
>Hi Netters,
>
>I need to re-amplify a PCR product, using the orignial primers for PCR
>with the gel purified product as template, because the yield of the 
>particular fragment is so low.
>
>Since I don't have much information about the DNA sequence and I can't 
>design the internal primers for re-amplication.
>
>What I got was always a smear even though I use as little as 0.1ng of my 
>purified product as template.
>
>Can anybody suggest me how to do it?
>
>Any recommendation is appreciate!
>
>Nelson.
>BHWFDUNG at cphkvx.cphk.hk

Subject: Re: re-amplication of PCR product without internal primers
From: Hong Dang, hdang at channel.neusc.bcm.tmc.edu
Date: 19 Aug 1994 21:01:34 GMT
In article <3336je$ogf at gazette.bcm.tmc.edu> Hong Dang,
hdang at channel.neusc.bcm.tmc.edu writes:
>In article <01HG3ABNW3QQ8WXHAF at cphkvx.cphk.hk>, BHWFDUNG at CPHKVX.CPHK.HK
writes:
>|> Hi Netters,
>|> 
>|> I need to re-amplify a PCR product, using the orignial primers for PCR
>|> with the gel purified product as template, because the yield of the 
>|> particular fragment is so low.
>|> 
>|> Since I don't have much information about the DNA sequence and I
can't 
>|> design the internal primers for re-amplication.
>|> 
>|> What I got was always a smear even though I use as little as 0.1ng of
my 
>|> purified product as template.
>|> 
>|> Can anybody suggest me how to do it?
>|> 
>|> Any recommendation is appreciate!
>|> 
>|> Nelson.
>|> BHWFDUNG at cphkvx.cphk.hk
>
>How did you purify your fragment? Did you get rid of EtBr?
>I tried it once with DNA out of gel containing EtBr, and got a smear.
>
>Hong
****

Use less! A typical reaction for us is to 
				1) do first pcr
				2) run about 10 uL (of the 50) on a 1-2% gel (with Ethidium in it)
				3) Cut out the band, using razor blade or yellow tip
				4) place band in 50 uL of water
				5) Heat 95oC for 5-10 minutes or leave ON at 4oC
				6)  Re-amp 0.01, 0.1 or 1 uL of that solution
				7) Almost always we get excellent yields using the 0.01 and 0.1
amounts
 We have found no problem with the small amnts of EThidium that are still
present.


Doug feinstein 
dlfeins at cumc.cornell.edu



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