Dear all,
I would like to ask for general recommendations to anyone with experience of using the aforementioned kit for their RACE PCR.
I am currently having difficulties on using the kit for both 5' and 3' RACE of which I am running out of options on where to troubleshoot, and hence any alternative point of view which I may have missed would be most welcomed.
At the moment, I have a total RNA sample that I know contains my mRNA of interest, as using the same sample, I managed to synthesize first strand cDNAs. To my understanding, since I used oligo dT primers to do so, the synthesized sequence should be originating from only mRNAs present within. With use of designed degenerate primers on the cDNAs, I managed to obtain a partial fragment of my gene of interest some 700bp long of which I used to design gene specific primers for RACE.
Unfortunately, after following the protocols outlined from first strand synthesis to nested RACE PCR, I have so far been unable to obtain any bands of any kind. All this is in view of using the same RNA aliquot obtained earlier, with integrity and concentration prior to use verified via denaturing gel electrophoresis via nanodrop. The primers I designed (sense for 3' RACE and antisense for 5' RACE) have been deemed to be good and later confirmed via amplification of the correct size of band.
From this result, and assuming the other components have been administered as sufficient, my main hunch as to where it could go wrong is on the first strand synthesis for both strands, of which the 5' CDS and 3' CDS primers (at 0.5µM per reaction,functions as oligo dT primer shall function) may have difficulty in binding to the polyA tails due to the initial total RNA concentration being low, but again I iterate that this is all a hunch and nothing is certain.
Should anyone else out there be familiar with RACE PCR of a different kind, attached with this email is the User Manual for the aforementioned kit, which may prove useful for comparison. Any help is much appreciated and I extend my thanks in advance for all the kind souls out there willing to do so :)
Hi Mohamad,
I used the Clontech SMART 5' RACE kit back in 2008 (See the Berg et al 2009, page 387). But before I attempt to help you, have you approached the Clontech support?
Best,
Rahmi
Article The expression of recombinant genes in Escherichia coli can ...
I use the same kit for 5' RACE and I got the result. what I do is follow the exact protocol and changes et al and I use every thing from the kit even the deionized water.
Main thing is the integrity of mRNA used, for that you can check your mRNa either by formaldehyde denaturing gel for RNA detection or you can incubate a little portion of your mRNa at 37C for 2 hours and remaining in -80 and then compare both mRNA (37 and -80) by using 1.5% EtBr gel. If the expression is different after 37C incubation that means your RNA contains RNase which is a huge problem. then you should be more careful during RNA extraction
Good luck
If you still need assistance, let me know. I have been troubleshooting the kit this spring with my two graduate students. We are trying to isolate two sRNAs from bacteria using this kit. We have used high quality RNA that is approximately 1ug/ul. We ordered a polyA polymerase to polyadenylate the RNA so we could use the 5' CDS and 3'CDS oligos provided by the kit to reverse transcribe "all" of the transcripts. One student also used a transcript specific oligo to try and target his transcript.
We took the cDNA generated in these reactions and used them with a GSP and their UPM (that anneals to the 5' end of the transcripts). Here we ended up with a mixture of bands they suggest cutting out and purifying. Well the question was, which one...and the kits has a limited number of reactions...and is super expensive.
MINE IS NOT AN ANSWER BUT I WILL LIKE TO ASK ANDREA, SINE HE HAS TROUBLESHOOTED THE KIT, DID YOU CHANGE YOUR ANNEALING TEMP. I USED THE 68OC PROVIDED, BUT HAD MULTIPLE BANDS, MAYBE I SHOULD CHANGE TO 65 AND DO 35 CYCLES? IF YOU CAN HELP OUT KINDLY LET ME KNOW
I used the SMARTer RACE 5'/3' kit for gene cloning. Worked well. Multiple bands in 5' race but gel extraction / purification led to recovery of full length transcript of my candidate gene.
Does anyone know why the kit recommends using UPM and GSP alone control PCR reactions?
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