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trouble with pcr - (Nov/08/2009 )

Hi all

Im in a confusion. Im trying to isolate a gene of 1.4kb . I isolated mRNA using phenol/sds method and in denaturation gel i saw 2 rRNA bands
then I performed RT-PCR and tried to ampllify it by pcr. But i am getting a band of 20000bp instead of 1.4kb . I donot understand it.Is it from genomic dna amplification?

Thanks in advance.

-deespike-

deespike on Nov 9 2009, 06:44 AM said:

Hi all

Im in a confusion. Im trying to isolate a gene of 1.4kb . I isolated mRNA using phenol/sds method and in denaturation gel i saw 2 rRNA bands
then I performed RT-PCR and tried to ampllify it by pcr. But i am getting a band of 20000bp instead of 1.4kb . I donot understand it.Is it from genomic dna amplification?

Thanks in advance.


hi Dee, do you RNAse treat your cDNA after reverse transcription to remove gDNA?

-DrRob-

DrRob on Nov 9 2009, 04:40 AM said:

deespike on Nov 9 2009, 06:44 AM said:

Hi all

Im in a confusion. Im trying to isolate a gene of 1.4kb . I isolated mRNA using phenol/sds method and in denaturation gel i saw 2 rRNA bands
then I performed RT-PCR and tried to ampllify it by pcr. But i am getting a band of 20000bp instead of 1.4kb . I donot understand it.Is it from genomic dna amplification?

Thanks in advance.


hi Dee, do you RNAse treat your cDNA after reverse transcription to remove gDNA?


no i dnt use dnase enzyme.im nt having it.

-deespike-

run a RT-PCR negative control first,where you only have your RNA and not your cDNA and then do a PCR for your control gene/gene of interest....if you get a product in this also(-RT), then its definitely genomic DNA contamination of your RNA.

-Helios-

Helios on Nov 10 2009, 09:30 PM said:

run a RT-PCR negative control first,where you only have your RNA and not your cDNA and then do a PCR for your control gene/gene of interest....if you get a product in this also(-RT), then its definitely genomic DNA contamination of your RNA.

Thank you for your suggestion .I shall be doing this and im eager to see the result.

-deespike-

Helios on Nov 10 2009, 09:30 PM said:

run a RT-PCR negative control first,where you only have your RNA and not your cDNA and then do a PCR for your control gene/gene of interest....if you get a product in this also(-RT), then its definitely genomic DNA contamination of your RNA.


hi

i peerformed pcr by putting negative and positive control to check the integrity of the cdna and mrna.I used GADPH primer for cdna and got amlification at the desired location. Further i got amplification of my desired gene with one of the primer set that i have designed without restriction sites involved in it.
But i donot get amplification with the other set of primers which have restriction enzyme sites at the 5'end for the same gene.I have varied annealing temperature but still im nt getting amplification.

What may be the possible causes for primers not giving amplification?

-deespike-

Hi Deespike,

How many restriction sites have you inserted, and what size primers? Perhaps you have reduced the specificity of your primers by adding too many non-specific bases.

-lab rat-

lab rat on Nov 12 2009, 12:48 PM said:

Hi Deespike,

How many restriction sites have you inserted, and what size primers? Perhaps you have reduced the specificity of your primers by adding too many non-specific bases.

hi

my forward primer is 27 bp in length and 18bp similarity with the gene sequence is there and rest includes restriction sites at the 5' ends .my reverse primer is 21bp length doesnot have any restriction sites but to increase the tm value and gc content i had added gc at the 5' ends.
The Tm value is 67 C for both the primers.
These primers are having some binding problem may be the annealing temp is getting higher than required.

-deespike-

lab rat on Nov 12 2009, 12:48 PM said:

Hi Deespike,

How many restriction sites have you inserted, and what size primers? Perhaps you have reduced the specificity of your primers by adding too many non-specific bases.

hi

my forward primer is 27 bp in length and 18bp similarity with the gene sequence is there and rest includes restriction sites at the 5' ends .my reverse primer is 21bp length doesnot have any restriction sites but to increase the tm value and gc content i had added gc at the 5' ends.
The Tm value is 67 C for both the primers.
These primers are having some binding problem may be the annealing temp is getting higher than required.

-deespike-