3 replies to this topic

[Madwelshboy]

Hi, I've been running a PCR to demonstrate the the expression of various genes in the placenta, in order to then use the placenta as a positive control for other tissues. Based on the literature, I know these genes are there and are highly expressed. However when i run my PCR, using the same primer sequences and conditions as have previously been reported, im getting a very poor yeild for my product.

The program is as follows:
95 C for 3.30min

For 40 cycles:
95 C for 30sec
Tm (55 C) for 30sec
72 C for 30 sec
55 C for 60sec

finaly, 72 C for 10min

The protocol is as follows:
1ul cDNA
2ul Buffer
0.5 dNTP
1ul x 2 primers
1.2ul MgCl2
0.2ul Taq
13.1ul dH2O

I've tried a temp gradient, a Mg Gradient, an increase template concentration and a increased primer concentration. So now i really dont know what else to try.

Any suggestions?

Edited by Madwelshboy, 04 June 2010 - 04:54 AM.

[HomeBrew]

Hi Madwelshboy -- welcome to the BioForums!

I can think of a couple of things to try.  First, increase the amount of Taq a bit (can you accurately measure 0.2 ul?)  Perhaps you could make a 10X stock of Taq in a larger volume and add 2 ul of that.  BTW, your reaction volume as listed only adds up to 19.5 ul -- I presume you were shooting for 20 ul?

Secondly, I've often found that published primers don't work as well as ones I've designed myself using a program such as Primer3.  Maybe you should design your own primers, perhaps by tweaking those you're using?

What are the sequences of the primers you're using?  How big a product are they supposed to produce?

[Madwelshboy]

HomeBrew, on Jun 4 2010, 01:34 PM, said:

Hi Madwelshboy -- welcome to the BioForums!

I can think of a couple of things to try.  First, increase the amount of Taq a bit (can you accurately measure 0.2 ul?)  Perhaps you could make a 10X stock of Taq in a larger volume and add 2 ul of that.

  

Its 0.2ul for each single reaction, at the very least i do 4 reactions.

HomeBrew, on Jun 4 2010, 01:34 PM, said:

BTW, your reaction volume as listed only adds up to 19.5 ul -- I presume you were shooting for 20 ul?

Opps left out the 0.5ul of dNTP in my orignial post (now edited)

HomeBrew, on Jun 4 2010, 01:34 PM, said:

Secondly, I've often found that published primers don't work as well as ones I've designed myself using a program such as Primer3.  Maybe you should design your own primers, perhaps by tweaking those you're using?

The problem with this is that, the primer sequences were previously published by my research group and that initial paper has then been expanded into what is now my phd. So my supervisor wants to stick with the same primers.

HomeBrew, on Jun 4 2010, 01:34 PM, said:

What are the sequences of the primers you're using?  How big a product are they supposed to produce?

See the attachment

Attached Thumbnails

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[HomeBrew]

Madwelshboy, on Jun 4 2010, 09:06 AM, said:

Its 0.2ul for each single reaction, at the very least i do 4 reactions.

OK, so you make a master mix, measuring 0.8 ul and aliquoting it into four 20 ul reactions?  It stills seems to be a small amount of Taq per reaction -- have you tried more per reaction?

Madwelshboy, on Jun 4 2010, 09:06 AM, said:

Opps left out the 0.5ul of dNTP in my orignial post (now edited)

What concentration is your dNTP mix at?  My concern is that you've got a limiting reagent in your reaction somewhere because you're cutting something too fine -- if there's not enough Taq or dNTPs, or primers, then once they're used up making product (well, the Taq doesn't get used up, but it does have a half life), it's game over.

You should be using something like 0.5-2.0 units Taq, 200 μM of each dNTP, and 0.2-0.5 μM each primer.  Try a reaction which uses the upper end of these ranges for each component, and see if your yield improves.

Madwelshboy, on Jun 4 2010, 09:06 AM, said:

The problem with this is that, the primer sequences were previously published by my research group and that initial paper has then been expanded into what is now my phd. So my supervisor wants to stick with the same primers.

I see.  And did you have a new set of these primers synthesized, or are you using old stock?