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
