PCR primers + large mRNA amplification - (Oct/02/2008 )
I am hoping an expert on PCR could shed some light to my questions
- I am designing PCR primers from pubmed nucleotide mRNA sequences. I choose my primers from the CDS part of the sequence... Is this the correct way to do it?
- I will need to amplify 1.7kb long mRNA sequences. Will a standard PCR reaction mix work? I have used this with success with small 200-700 bp fragments.
Can anyone help?
Thank you Eleni
- I am designing PCR primers from pubmed nucleotide mRNA sequences. I choose my primers from the CDS part of the sequence... Is this the correct way to do it?
- I will need to amplify 1.7kb long mRNA sequences. Will a standard PCR reaction mix work? I have used this with success with small 200-700 bp fragments.
Can anyone help?
Thank you Eleni
As long as the source of your DNA template is the same as the one you used to design your primer from. and youre not amplifying mRNA in this case, it would be just DNA.
1.7kb should be easy . i have done 2.7kb b4 lol no problem.
Just make sure the template DNA are of good quality.
if there's any problem we'll help.
- I am designing PCR primers from pubmed nucleotide mRNA sequences. I choose my primers from the CDS part of the sequence... Is this the correct way to do it?
- I will need to amplify 1.7kb long mRNA sequences. Will a standard PCR reaction mix work? I have used this with success with small 200-700 bp fragments.
Can anyone help?
Thank you Eleni
As long as the source of your DNA template is the same as the one you used to design your primer from. and youre not amplifying mRNA in this case, it would be just DNA.
1.7kb should be easy . i have done 2.7kb b4 lol no problem.
Just make sure the template DNA are of good quality.
if there's any problem we'll help.
I am not sure I understand your reply
I will be doing RT-PCR for mRNA and want to design primers to an mRNA sequence for the PCR. I choose my primers from the CDS part of the mRNA sequence is this correct?
What do you whant to amplify DNA or cDNA ???and whats the purpose???
What I do is take the complete sequence and highlight the exons (is tedious but no matter if need gDNA or cDNA you have it handy).
If what to distinguish between gDNA and cDNA design primers that is in 2 consecutive exons. There will be a difference in size (gDNA is bigger because of intron sequence).
If going to do a QPCR of cDNA use a primer set in the exon and no bigger than 200bp.
If looking for polymorphism use gDNA secuence.
Well possibles are endless so telling what is you template and purpose will be easier to give advise.
What I do is take the complete sequence and highlight the exons (is tedious but no matter if need gDNA or cDNA you have it handy).
If what to distinguish between gDNA and cDNA design primers that is in 2 consecutive exons. There will be a difference in size (gDNA is bigger because of intron sequence).
If going to do a QPCR of cDNA use a primer set in the exon and no bigger than 200bp.
If looking for polymorphism use gDNA secuence.
Well possibles are endless so telling what is you template and purpose will be easier to give advise.
I am trying to amplify cDNA that I have created from mRNA by Reverse Transcription. Basic RT-PCR.
I will DNase treat my cDNA so I am not so bothered about distinguising gDNA from cDNA
I just need PCR primers from the coding region of the mRNA, and I chose to take the CDS part of the mRNA sequence (I think that I used to do that a few years ago but not sure) and not from the exon sequenses (although thats the logical thing to do). I thought that CDS in the pubmed mRNA sequence means coding sequence and thats why I chose my primers from that sequence. Did I help at all?
To be honest i have never done PCR from this template. but designing primer based on the mRNA is probably the only way to go for this procedure unless you want to amplify the gene that hasn't undergone gene splicing.
5' PRIMER F---------------------------> 3'
3' cDNA NNNNNNNNNNNNNNNNNNNTTTTTTTT5'
<-------------------------------PrimerR 5'
Is that kinda like what you're planning to do ?