hello, you can try to clone the PCR product you got from gel in any cloning vector like
pJet or pGem, since they require low amounts of PCR products, then you can digest the vector and get the fragment ready for further cloning. it is two steps more but may help you good luck FH ________________________________ From: DK <[email protected]> To: [email protected] Sent: Sun, December 26, 2010 12:28:15 AM Subject: Re: gel elution of 6KB PCR product In article <[email protected]>, "B.Rama chandran" <[email protected]> wrote: >Dear All, > > I am facing problem in the gel elution of 6KB PCR product, I am >getting very less yield (~10ng/micto liter). I am using Sigma gel >extraction kit (catalog no:NA1111). Since yield is very less I couldn't use >that product for restriction digestion. If you have any suggestion please >give me. If you know any other technique which will give better yield please >let me know that. What is the yield as % of input? If you didn't have much to begin with, nothing can help. ~50% on gel-extraction are ~ normal. If it is the yield that is poor, try the same protocol with 2 kbp. If the result is good, your kit is not appropriate for 6K. If the result is bad, either your reagents are gone bad/improperly prepared or you are doing something wrong. I've purified 14 kbp from gel with about 30% using Qiagen's kit. Never a problem. DK _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods --0-1362310605-1293367365=:9174 Content-Type: text/html; charset=us-ascii <html><head><style type="text/css"><!-- DIV {margin:0px;} --></style></head><body><div style="font-family:arial,helvetica,sans-serif;font-size:12pt">you can try to clone the PCR product you got from gel in any cloning vector like pJet or pGem, since they requier low amounts of PCR products, then you can digest the vector and get the fragment ready for further cloning. it is two steps more but may help you<br>good luck<br>FH<br><br><div style="font-family: arial,helvetica,sans-serif; font-size: 12pt;"><div style="font-family: arial,helvetica,sans-serif; font-size: 12pt;"><font face="Tahoma" size="2"><hr size="1"><b><span style="font-weight: bold;">From:</span></b> DK <[email protected]><br><b><span style="font-weight: bold;">To:</span></b> [email protected]<br><b><span style="font-weight: bold;">Sent:</span></b> Sun, December 26, 2010 12:28:15 AM<br><b><span style="font-weight: bold;">Subject:</span></b> Re: gel elution of 6KB PCR product<br></font><br> In article <<a ymailto="mailto:[email protected]"; href="mailto:[email protected]";>[email protected]</a>>, "B.Rama chandran" <<a ymailto="mailto:[email protected]"; href="mailto:[email protected]";>[email protected]</a>> wrote:<br>>Dear All,<br>><br>> I am facing problem in the gel elution of 6KB PCR product, I am<br>>getting very less yield (~10ng/micto liter). I am using Sigma gel<br>>extraction kit (catalog no:NA1111). Since yield is very less I couldn't use<br>>that product for restriction digestion. If you have any suggestion please<br>>give me. If you know any other technique which will give better yield please<br>>let me know that.<br><br>What is the yield as % of input? If you didn't have much to begin <br>with, nothing can help. ~50% on gel-extraction are ~ normal. If it is the <br>yield that is poor, try the same protocol with 2 kbp. If the result is good, <br>your kit is not appropriate for 6K. If the result is bad, either your <br>reagents are gone bad/improperly prepared or you are doing something <br>wrong. I've purified 14 kbp from gel with about 30% using Qiagen's <br>kit. Never a problem. <br><br>DK<br>_______________________________________________<br>Methods mailing list<br><a ymailto="mailto:[email protected]"; href="mailto:[email protected]";>[email protected]</a><br><span><a target="_blank" href="http://www.bio.net/biomail/listinfo/methods";>http://www.bio.net/biomail/listinfo/methods</a></span><br></div></div> </div><br> </body></html> --0-1362310605-1293367365=:9174-- _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
