Noi/NOTES/2013-3-14

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Amplification TEST[edit]

  • There are two set of primers
  • Set 1: 1 forward primer without barcode && 3 different reverse primers with different barcodes (Indx1-3)
1-1. gDNA (12878) Indx1
2-1. Total RNA Indx2
3-1. ds cDNA no RNasA treatment Indx3
4-1. ds cDNA RNaseA treatment Indx3
  • Set 2: 3 different forward primers with different barcodes && combination of 9 reverse primer without barcodes
1-2. gDNA (12878) Indx1
2-2. Total RNA Indx2
3-2. ds cDNA no RNasA treatment Indx3
4-2. ds cDNA RNaseA treatment Indx3
  • Note: Since we have only 3 different indexes, and the two set of indexes are the same, the libraries amplified by the two sets of primers can not be pooled together and sample 3-1 and 3-2 will not combinded in the pooled

PCR TEST set up[edit]

Set 1: by Matt[edit]

Sample set 1 Index Primer names xxxxxxxxxxxxxxxxx
1-1. gDNA (12878) Indx1
2-1. Total RNA Indx2
3-1. ds cDNA no RNasA treatment Indx3
4-1. ds cDNA RNaseA treatment Indx3

PCR Mix

Components 1 rxn 5.5 rxn mix
Captured template 1.00 0.00
10uM Forward no barcodes 0.40 2.20
10uM Reverse +Indx 0.40 0.00
2x KAPA SYBG fast MM 12.50 68.75
H2O 10.70 58.85
Total 25.00 137.50
  • Aliqout 23.6ul, add 0.4ul of reverese primer, add 1ul of circular DNA

Set 2 : by Noi[edit]

Sample set 2 Index Primer names xxxxxxxxxxxxxxxxx
1-2. gDNA (12878) Indx1
2-2. Total RNA Indx2
3-2. ds cDNA no RNasA treatment Indx3
4-2. ds cDNA RNaseA treatment Indx3

PCR Mix

Components 1 rxn 5.5 rxn mix
Captured template 1.00 0.00
10uM Forward + Indx 0.40 0.00
10uM Reverse no barcodes 0.40 2.20
2x KAPA SYBG fast MM 12.50 68.75
H2O 10.70 58.85
Total 25.00 137.50
  • Program

98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min

Set 1[edit]

Components 1rxn 6.5 rxn
Captured template 12.00 0.00
10uM Forward no barcodes 2.00 13.00
10uM Reverse +Indx 2.00 0.00
2x KAPA SYBG fast MM 50.00 325.00
H2O 34.00 221.00
Total 100.00 650.00
  • Aliquot 86ul, add 2ul of reverse Ind, 12ul of circular DNA
  • Will add more for second trial by Matt for set 2 experiment (use 10uM and 100uM of reverse primers

Set 2[edit]

Components ' '
Captured template 1rxn 6.5 rxn
10uM Forward + Indx 12.00 0.00
100uM Reverse no barcodes 2.00 0.00
2x KAPA SYBG fast MM 2.00 13.00
H2O 50.00 325.00
Total 34.00 221.00
100.00 650.00
  • Aliquot 86ul, add 2ul of Forward Ind, 12ul of circular DNA
  • Program

98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x13 -> 72C 3min (total 21 cycles)

  • Perform bead purification , set 1 elute total volume 60ul, set 2 elute total volume 50ul.
  • Verify by PAGE an do PAGE quantification to see the ratio of each product before pooling the libraries.
  • Note: I quantified based on the expected band ~250bp and ~400 (two copy of target based on amplification of circular DNA.

File:ZhangLab 2 2013-03-15 11hr 27min SeqLib Insitu.jpg

Samples Conc. (ng/ul) Yields(ng) Pooled volume(ul)
gDNA-set1 26.50 1590.05 37.73
dsDNA -RNaseA set1 34.14 2048.43 29.28
dsDNA +RNaseA set1 33.33 1999.55 30.00
gDNA-set2 27.11 1355.44 10.41
dsDNA -RNaseA set2 7.05 352.73 40.00
dsDNA +RNaseA set2 10.38 519.25 27.17