Noi/NOTES/2013-3-14
Jump to navigation
Jump to search
- [Link to calendar]
- Continued from: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2013-3-12
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 |