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S   0$ `` [P ]*    0 `  [P _*    0 `  [P _*  H  0޽h ? ̙33 $Blank PresentationN ( B  B  <De/|t___PPT9VN JIdentify difficult sequence contexts polyN tracts, high GC Select unique primers within your gene of interest Watch out for repetitive sequence Be aware of pseudogenes gene AGGCTTAGCCCCAGTAGGGA pseudo AGGCTTAGCCTCAGTAGGGA *All individuals heterozygous&?#[&?#  3HL&    d  Y  @`  C bAJ\\192.168.85.1\mrieder\snapshot1.png@   <D0 ^PCR primer selection caveats    <$  <  H  0޽h ? ̙33 0 @( PS9    <sP} VPCR Primer Selection  >  <O Z  PCR_Overlap - Primer3 (MIT) primer selection software Selection settings: 23 mers, Tm = 60 deg C eLONGase PCR Kit (Gibco Life Technologies) Amplicon Size: 900-1000 bp Amplicon Overlap: 150-250 ~ 40% redundancy for sequencing and SNP verification Screen for repetitive elements, sequence context which reduce sequencing read length. (e.g. microsatellites, poly N tracts). Verify uniqueness of primers in region/gene of interest or gene family 80-90% amplicons successful - first pass conditions 2nd round of selection or optimization (i.e. annealing temperature) nearly 100% success 10-15% PCR failure rate during production (random dropouts)X8AFIS              e    H  0޽h ? ̙33l    @$ (  $F d1 $ d, $ C A proci.bmp 00000754Big Mark B03A5E64:d1 N @   $ @  B $ ZDo@  B $ ZDo 0 B $ ZDop  B $ ZDo  B  $ ZDo @ B  $ ZDo P B  $ ZDo  B  $ ZDo  B  $ ZDo@ P B $ ZDo  # $ <N _  ;Ave. Amplicon Size = 980 bp Ave. Amplicon Overlap = 160 bp X            $ <N` APROCI (Protein C Inhibitor)  H $ 0޽h ? ̙33 P(V( HB ( ( <Hy  VPCR primer selection  T ( <4QtC/___PPT9  Type: >pcr_overlap.pl  file gene.ncbi.reference.fasta  gene gene The program will begin selecting overlapping primers. A report will go to the screen. Picking primer set 1.. Picking primer set 2.., etc The program will finish picking primers  then type: >gvim gene.primers.txt This file will contain information about the primers which were selected. Example: ttctgtaaatgccacgtttcc 59.989 # Forward primer and melting temp # aaatttcggccttgctttg 60.195 # Reverse primer and melting temp # 2.2060 # Primer quality - (Primer3) # 6.00 4.00 # Left and right primer self-complementarity score (Primer3) # 4.00 4.00 # Primer pair complementarity and 3' complementarity score (Primer3) # 159 21 # Forward primer position (5') and length # 1018 19 # Reverse primer position (5') and length # 860,(overlap) # Product size (including primers) # Check the uniqueness of primers by typing: >cross_match gene.ncbi.reference.fasta gene.primers.fasta  minscore 15 > gene.primers.xm Make a picture of the primers that were selected: >DrawMap  beg 0  end (size of ref seq)  scale 250  list gene2.drawmap.list  outfile gene2.ps View the picture you primer layout, type: >ps2pdf gene2.ps >acroread gene2.pdf 65! 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