Help: PCR Amplification Problems

Christopher T. Huhta huhta at euripides.ccs.itd.umich.edu
Mon Jul 12 13:55:36 EST 1993


I am currently stuck in the process of amplifing a VERY RARE message in a
human T-cell line.  This message, like a said is very rare, but we have
successfully amplified it once before.  In general, I need help optimizing
my PCR, and as I am fairly new to PCR techniques, I was wondering if you
all could ehlp me out.  Here's the protocol I am using withou success
right now:

I.  RT

1.  Prepare RT Master Mix

To each tube...
4 ul Gibco BRL First strand buffer mix
2 ul 0.1 mM DTT
1 ul of each dNTP
0.5 ul RNAse inhibitor

Total : 10.5 ul

2.  In separate tubes preepare RNA

1 ug mRNA from T-cells (2.0 ul volume)
0.6 ug Random Primers (2.5 ul @ 0.2 ug.ul)
3.5 ul DEPC H2O

Total 8.0 ul

3.  Incubate tubes containig mRNA at 70C for 10 min.  Immediatelt put on ice.
4.  Add 10.5 ul RT Mix to each tube.
5.  Incubate at 37C for 23 min and remove to room temp.
6.  Add 1 ul M-MLV RT (Gibco) to each tube.  (200 U/ul?)
7.  Run profile.  37C for 60 min, 99C for 10 min, hold at 4C


II.  PCR


1.  Prepare PCR Master Mix

To each tube..

.75 ul 1500 mM Kcl
.4  ul 1000 mM Tris Cl pH 8.0
.25 ul TAQ Poly
1   ul of each dNTP (10 mM stock)
2.5 ul MgCl2 (25 mM stock)
24.5 ul  H2O

Total volume 31.4 ul per tube

2.  To each tube add 2.0 ul of 5' and 3' primers (.25 ug/ul stock)
3.  Add 2.0 ul of appropriate cDNA (form 20 ul RT volume)
4.  Mix, spin briefly, and cover with mineral oil.
5.  Run Profile.

    94C for 1:15, 55C for 2:15, 72 for 3:20
    40 Cycles
    Hold at 4C


The piece I am trying to amplify is 1.7 kb in length.  ANy help concerning
optimizing this would be appreciated including references to where I can
look for optimization techniques.  I have run positive control (beta
actin) in these preps and have gotten nice strong positive control, but no
1.7 kb fragment.  I have tried other primers and checked the original
primers for primer dimers etc. to no avail.  I am completely stuck unless
I can troubleshoot this experiment....HELP!

Thanks for taking the time to wade through this.

Chris Huhta
Dept. of Internal Medicine
Univ. of Michigan
huhta at euripides.ccs.itd.umich.edu

Again, thanks.



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