Difficulties getting a 4 kb PCR product - (Sep/21/2006 )

Dear colleges:
We are struggling with a PCR! We would greatly appreciate to get your opinions and suggestions regarding our approach.

We need to amplify a 4 kb DNA fragment. The template is a cDNA synthesized by reverse transcription (RT) from total cellular (eukaryote) RNA (random primers).
The RNA is always purified from DNA contamination (Dnase plus Qiagen column). The RT is done using 1 ug of total purified RNA. The cDNA is cleaned up in Quiquick-PCR purification columns-Qiagen and then 2 ul is used for PCR.
The PCR cycle is as follow:
94C, 2 min
(94C, 15 s; 60C, 30 sec) x 30
68C, 4 min

We tested:
a- polymerases, first we tried Phusion-Finnzymes and then decided to go with Platinun Pfx-Invitrogen

b- gradient temperatures 5 +/- around MT with no products at any

c- reverse transcriptase first used Promega kit (AMV RT), now we decided to use Superscript II-Invitrogen

d- reverse transcription reaction time from 15 min to 1 h (with Promega kit, need to try with new enzyme)

We found out that increasing the reverse transcription time probably generated better templates because the last PCRs (at one of the gradient temperature Rxs) we got several bands with the longer is around 1 kb.

Yet, we are far from a 4 kb product!
Should we consider to use RACE technology, too?

We would be very grateful for your input. Thanks in advance!


Nancy

hmmmm

why no extension step during your PCR?

have you tried gene-specific primers for the RT, in order to generate fewer truncated forms?

As others wrote before, extension is missing . Is 68C the extension ?

We used superscript III for RT (60min) and used to use 1 ul of the reaction for PCR without cleaning. It worked quite efficiently.

uuppsss!!!
My mistake! Sorry...!
The cycle is as follow:

94C, 2 min
(94C, 15 sec; 60C, 30 sec; 68C, 4 min) x 30
68C, 10 min




Thanks!!!

No, we have not used gene specific primers during the RT. But, it is a good idea!

And, we ordered Superscript II! If things keep not working we may have to get III.


Nancy

For the Phusion from Finnzymes, there is no need to drop the extention temperature.

Keep it at 72 Celcius and drop the extention time to 2mins.

How are the primer? What is their Tm? Could we view their sequence?