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To: Multiple recipients of list HUM-MOLGEN <HUM-MOLGEN@NIC.SURFNET.NL>
Subject: BIOT: diff. PCR express. and repl./protocols PCR genotyping
From: Arthur Bergen <bergen@AMC.UVA.NL>
Date: Sat, 27 May 1995 12:51:59 +0100

Note from the editor:

This BIOT message contains 4 submessages:

1) CALL for technical help -- differential PCR
2) REPLIES to PCR genotyping -- 3 different non-radioactive protocols!


The BIOT TOPIC of HUM-MOLGEN is open for collaborative technical help,
tips for new products, techniques and protocols, semi-commercial messages
about new techniques, apparatus and products. BIOT is especially a
contact forum between the academic and the commercial world. Offering and
finding of Jobs are partly covered by BIOT and partly by ANNO.
Communication is FREE of charge, fast and in between THOUSAND
HUM-MOLGENeticist all over the world!

Best wishes,

BIOT Editors

Martin Kennedy
Arthur Bergen


**************************************************************************
1) CALL:

Please, could you inform us, if
ANYBODY has had GOOD EXPERIENCE WITH DIFFERENTIAL EXPRESSIN OF pcr WE ARE
WORKING ON IT FOR 2 YEARS AND MOST OF OUR "DIFFERENTIAL EXPRESSED BANDS"
ARE ARTFACTS; Thanks

Yacob Weinstein
Head Dep. of Microbiol. & Immunol.
Health Sciences - Ben Gurion Univ. Beer Sheva ISRAEL 84105
EMAIL yacob@bgumail.bgu.ac.il
Phone 972 7 400858 FAX 972 7 277453
**************************************************************************

2-4: REPLIES to PCR genotyping

I've been doing Ag staining for sometime now and thought you might
appreciate the following info. :

Gels: Use non-denaturing gels of 1mm thickness for ease of handling and gels thinner than 1mm are prone to the silver being washed out prior to the developing step.

Resolution: CA repeats varying in size from 100-250bp in size can be
easily resolved on 7-9% acrylamide gels. Use ready-made Accugel (National
Diagnostics 19:1 Sequencing grade) (40%) for best results.

Samples: Mix samples with a sucrose loading buffer as glycerol reacts witth
borate in TBE running buffers and distorts the migration of DNAs.

Electrophoresis: This depends on the % of acrylamide used but generally I
have been running the gels at a low voltages of 100-140V. I normally do a
4hr run at 100V of a 6% acrylamide gel till thebromophenol blue just
reaches the bottom to determine the degree of resolution required per
repeat and then do a longer run to obtain the degree of resolution required

Photography: Silver-stained gels can be photographed relatively easily
using a light box and camera as done for protein gels.

Interpretation: Generally if you have a series of bands below a densely-
stained band, these are stutter bands of a lower intensity than the real
band. Allele-drop out occurs when the smaller allele amplifies more
efficiently than the larger allele - it is necessary to be aware of this.

Tips for consistently good results: fill the bottom tank with the minimum
quantity of buffer, this ensures straight electrophoresis across the gel
- keep sample volumes small, large volumes lead to loss of resolution (10-
15ul is fine)
- wear clean gloves when pouring off AgNo3 solution
- store AgNO3 in clear bottles but away from light so that precipitates
can be seen - these solutions can be used several times until precipitates
form
- keep supply of staining trays dedicated for Ag staining - any contact
with alkali during staining will mark the gel
- keep the rounds of PCR to a minimum - gauge the number of cycles requireed
by doing controls to check for overamplification of bands as Ag staining
is very sensitive

Here then is the protocol I currently use which works very well:

1. Fix gel by incubating in 10% ethanol/0.5% acetic acid for 5mins.
2. Remove soution by vacuum.
3. Stain gel in 0.1% AgNO3 for 15 mins
4. Remove AgNO3 and keep for future use.
5  Pour on a solution of 1.5% NaOH/ 0.1% formaldehyde to develop the silver
   - takes about 5-10 mins depending on strength AgNO3 used.
Note: For ease of handling to photograph the gel leave the gel in a sand
wich box with one of the glass plates under the gel so that the gel can be easily lifted out of box.

Hope the above notes will be useful.

Dr Shirin Joseph

*************************************************************************

This message was originally  submitted by Tom_Frank@MAILQM.PDS.MED.UMICH.EDU to
the HUM-MOLGEN  list at NIC.SURFNET.NL.

.Hi
>I'm interested in doing PCR genotyping and would like some advice. I've
>previously done PCR genotyping of dinucleotide repeats using 32P
endlabeled
>primers which worked well but I'd like to explore non-radioactive
methods..

     I wonder if you have tried simply ethidium-bromide staining your
acrylamide gels? This is what we routinely use to see
microsatellite-derived PCR products amplified from one-fifth of the DNA
extracted from a paraffin-tissue section. If your starting material is
much less, however, this may not be sensitive enough. Our marker is the
old but readily available standby HaeIII-digested PhiX174 to make sure our
product is in the right ballpark, but running control DNA of known
polymorphism (CEPH DNA for example) would be better, if you can get it.
Good luck,
Tom Frank
tfrank@umich.edu



**************************************************************************
> I'm interested in doing PCR genotyping and would like some advice. I've
> previously done PCR genotyping of dinucleotide repeats using 32P endlabeled
> primers which worked well but I'd like to explore non-radioactive methods.
> Ag staining has been suggested and I'd be grateful for any useful tips on
> this.
> Also, I used to size the alleles using a sequencing ladder but I wonder if
> there's any other way. The PCR product I'm expecting would be abt 250 bp
> with 2bp differences between alleles.
> Thanks
> Agnes
>
There are other ways. We use a modification of the multiplex sequencing
method of George Church. Multiple PCR assays are run per lane of the
polyacrylamide gel, the gel is then blotted onto nylon membrane and
probed with a biotinylated-Strep-AP labelled oligo (one of the original
PCR primers). We use CSPD (recently started to use CDP-star with
promising results) as the substrate for the chemiluminescent reaction.
Once the bugs are ironed out this works pretty well. We are getting
between 16 and 24 assays per lane at present.


___________________________________________________________________________
Simon Foote,                            Laboratory of Mammalian Genetics
                                        Walter and Eliza Hall Institute
tel 61-3-345-2611                       P.O. Royal Melbourne Hospital
fax 61-3-347-0852                       Victoria 3050, Australia
___________________________________________________________________________


   
 
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