Noi/NOTES/2012-7-14
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epMotion test (AMPure bead purification)[edit]
- Continued from: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2012-7-12
- Things need to adjust
- Speed of aspiration and dispension of PCR product
- Avoid to reuse the tips to pipette the same reagent like EtOH in washing step. This need to adjust protocol to be a little more complicated in fill in the sample number in each step.
Plan of epMotion testing (two round AMPure bead purification and vary amount of start DNA amount)[edit]
- DNA amount range (in 80ul of the liquid)
- 5ng
- 10ng
- 25ng
- 50ng
- 100ng
- 200ng
- The pooled PCR product from Sergio's samples is approximately 22ng/ul by (PAGE quantification)
DNA amount (ng) | Total volume (ul) | Conc (ng/ul) | DNA (stock 22ng/ul) | H2O | Total volume | DNA amount (ng) |
5 | 80 | 0.0625 | 2.84 | 997.16 | 1000.00 | 5 |
10 | 80 | 0.1250 | 5.68 | 994.32 | 1000.00 | 10 |
25 | 80 | 0.3125 | 14.20 | 985.80 | 1000.00 | 25 |
50 | 80 | 0.6250 | 28.41 | 971.59 | 1000.00 | 50 |
100 | 80 | 1.2500 | 56.82 | 943.18 | 1000.00 | 100 |
200 | 80 | 2.5000 | 113.64 | 886.36 | 1000.00 | 200 |
Plate layout[edit]
' | 1 | 2 | 3 | 4 | 5 | 6 |
A | X | O | X | O | X | O |
B | O | X | O | X | O | X |
C | X | O | X | O | X | O |
D | O | X | O | X | O | X |
E | X | O | X | O | X | O |
F | O | X | O | X | O | X |
G | X | O | X | O | X | O |
H | O | X | O | X | O | X |
DNA amount in 80ul (ng) | 5ng | 10ng | 25ng | 50ng | 100ng | 200ng |
O: H2O, X: +DNA with different amount
Plan of contamination test[edit]
1) After adding DNA and H2O to each well, I'll take 2ul to test contamination before bead purification. This to make sure that there is no contamination. If there is some we can compare the level to after the 1st and 2nd round of purification
2) Take 5ul after the 1st purification, and in the last step just transfer keep DNA in the old plate.
3) Set up PCR reaction for before purification, 1st purification and 2nd purification and save in 4C (~ about 165 rxn, volume 25ul in each reaction)
PCR set up[edit]
Components | 1x | 170.00 |
DNA template | 2.00 | 0.00 |
100uM AmpFV6.3NH2 | 0.05 | 8.50 |
100uM AmpRV6.3NH2 | 0.05 | 8.50 |
2x KAPA | 12.50 | 2125.00 |
H2O | 10.40 | 1768.00 |
Total | 25.00 | 4250.00 |
98C 30sec (98C 10sec -> 58C 20sec-> 72C 20sec) x 35 cycles 72C 2min