HUM-MOLGEN archive: BIOT: diff. PCR express. and repl./protocols PCR genotyping

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 ___________________________________________________________________________