snip-SNP mapping with CB4856 polymorphisms

 

 

<center> <h1> snip-SNP mapping with CB4856 polymorphisms</h1> </center>

 

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Materials:

• Worm Lysis Buffer
• PCR reagents
• PCR primers flanking a SNP
• 2X restriction mix with the appropriate enzyme

Procedure:

1. 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
2. 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
3. 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
4. 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>