snip-SNP mapping with CB4856 polymorphisms
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<center> <h1> snip-SNP mapping with CB4856 polymorphisms</h1> </center>
Materials:
- Worm Lysis Buffer
- PCR reagents
- PCR primers flanking a SNP
- 2X restriction mix with the appropriate enzyme
Procedure:
-
Generate recombinant lines for the mutation of interest
- Mate your strain homozygous for the mutation of interest with the polymorphic strain CB4856
- Pick 6-8 F1 cross-progeny to individual plates
-
Pick F2 animals homozygous for your mutation to individual plates to establish lines
- For maternal-effect mutations, pick all F2s to individual plates and keep the ones showing the mutant phenotype
-
Prepare template DNA for SNP-analysis
- Pick mutant F2 animals individually into 20 ul H2O in PCR tubes
- Add 20 ul of 2X worm lysis buffer (w/ freshly added proteinase K) to each tube
- Process template as for Single Worm PCR
-
Amplify polymorphic region by PCR
- Aliqout 22.5 ul of PCR reaction mix to individual tubes
- Add 2.5 ul of template DNA to each
component
<center> <u>final conc.</u></center>
<center> <u>vol per rxn</u></center>
<center> <u>mix for 50 rxns</u></center>PCR Buffer
<center> 1 X</center>
<center> 2.5</center>
<center> 137.5</center>10X Buffer
dNTPs
<center> 100 uM / each</center>
<center> 0.1</center>
<center> 5.5</center>25 mM dNTPs
forward primer
<center> 0.5 uM</center>
<center> 0.625</center>
<center> 34.5</center>20 uM primer
reverse primer
<center> 0.5 uM</center>
<center> 0.625</center>
<center> 34.5</center>20 uM primer
template DNA
<center> (2.5)</center>
<center> --</center>DNA template
Taq Polymerase
<center> 0.5 - 1.0 U</center>
<center> 0.1</center>
<center> 5.5</center>Taq
<center> 18.55</center>
<center> 1021</center>H2 0
-
Run PCR reaction for 35 cycles with the following parameters:
- 10 secs @ 94 degrees, 10 secs @ 55 degrees, extension time (approx 30 secs/500bps) @ 72
-
Determine polymorphism carried by each recombinant worm using restriction digests
-
Add 8 ul 2X restriction mix to 8 ul PCR product (contains 50 mM KCl, 10 mM Tris 8.3, 1.5 mM MgCl2 )
- 2X restriction mix (volumes for 50 rxns)
<center> <u>ideal restriction enzyme buffer</u></center>
<center> <u>NEB#2</u></center>
<center> <u>NEB#3</u></center>component
<center> <u>stock soln</u></center>
<center> <u>conc in 2X</u></center>
<center> <u>vol of stock</u></center>
<center> <u>conc in 2X</u></center>
<center> <u>vol of stock</u></center>NaCl
<center> 5 M</center>
<center> 50 mM</center>
<center> 4.5 ul</center>
<center> 150 mM</center>
<center> 13.5 ul</center>Tris (pH7.5)
<center> 1 M</center>
<center> 10 mM</center>
<center> 4.5 ul</center>
<center> 10 mM</center>
<center> 4.5 ul</center>MgCl2
<center> 1 M</center>
<center> 18.5 mM</center>
<center> 8.3 ul</center>
<center> 18.5 mM</center>
<center> 8.3 ul</center>DTT
<center> .2 M</center>
<center> 2 mM</center>
<center> 4.5 ul</center>
<center> 2 mM</center>
<center> 4.5 ul</center>BSA
<center> 100X</center>
<center> 200 ng/ul</center>
<center> 9 ul</center>
<center> 200 ug/ul</center>
<center> 9 ul</center>enzyme
<center> 1 U</center>
<center> 45 U</center>
<center> 1 U</center>
<center> 45 U</center>H2 O
<center> 420 ul</center>
<center> 411 ul</center>
-
Add 8 ul 2X restriction mix to 8 ul PCR product (contains 50 mM KCl, 10 mM Tris 8.3, 1.5 mM MgCl2 )
