use PCR for cloning of long fragment??

Martin Offterdinger a8803349 at unet.univie.ac.at
Wed Oct 2 07:32:22 EST 1996


In article <51rcl0$c3i at info.service.rug.nl>, g.j.e.j.hooiveld at farm.rug.nl (Guido Hooiveld) says:
>
>Hello,
>
>As a novice starting to use PCR I am looking for some advice.
>
>I would like to clone a cDNA (4.3 kb) into a baculovirus expression vector. I 
>also want to include a histidine tag at the COOH-end of the protein allowing 
>affinity purification.
>However, the cDNA contains a 100 bp untranslated region at the 5'-end (and one
>at the 3'-end).
>
>First of all, is it necessary to introduce a new restriction site closer to 
>the 5'-end of the ORF (to get rid of the non-coding sequence like I have to
>do at the 3'-end) or has the 100 bp region no influence on the expression 
>level of the protein?
>
>Secondly, is it feasible to use PCR for amplifying a 4 kb sequence with high 
>fidelity? If this is possible which polymerase should I use (Taq, Pfu, Pwo)?
>
>Any help or literature references would be very appreciated!
>
>Please post here or send an e-mail directly to:
>g.j.e.j.hooiveld at farm.rug.nl
>
>Thanks in advance!
>
>Guido
>-- 
>***************************************************************************
>* Guido Hooiveld                              |                           *
>* Groningen University Center for Pharmacy    |phone: (+)31 50 3637565    *
>* dep. of Pharmacokinetics and Drug Delivery  |       (+)31 50 3633272    *
>* A. Deusinglaan 1                            |fax:   (+)31 50 3633247    *
>* 9713 AV Groningen                           |                           *
>* The Netherlands                             |                           *   
>***************************************************************************
>
Dear Guido!

You could use a kit from Perkin Elmer: XL RNA PCR Kit, I have used it to amplify 
a 2kB fragment from a cell line, without any mistakes. The Kit uses rTth Polymerase
in a one tube reaction to reverse transcribe and to amplify the target sequence.
It can be used for amplicons up to 5,6 kB.
Hope this helps                   Martin



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