Revision as of 21:03, 15 March 2013 by >Noi
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
|
98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min
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
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
|