Noi/NOTES/2012-5-14: Difference between revisions
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- Spin at 14,000rmp for 5min | - Spin at 14,000rmp for 5min | ||
- Dry the pellet in the hood and resuspend with 100ul H2O | - Dry the pellet in the hood and resuspend with 100ul H2O | ||
- Re-purified each amplicon with two Qiaquick columns, eluted with 40ul EB. | - Re-purified each amplicon with two Qiaquick columns, eluted with 40ul EB => total volume 120ul each. | ||
- Measure concentration. | - Measure concentration with Nanodrop. |
Revision as of 02:02, 15 May 2012
Probe production for the HiResChrPaint project
- Continued from the plan: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2012-5-8
Production PCR
- V4 pool for all four sets (12 reaction for each set)
Components | Volume (ul) | 49x rxn mix |
20nM oligo pool | 0.20 | 0.00 |
2x Kapa SYBG qPCR MM | 50.00 | 2,450.00 |
100uM pAP1V41U | 0.40 | 19.60 |
100uM AP2V4 | 0.40 | 19.60 |
2mM aa-dUTP | 3.00 | 147.00 |
H2O | 46.00 | 2,254.00 |
Total volume (ul) | 100.00 | 4,900.00 |
- Aliquot 1197.6 ul in 1.5 mL tube, add 2.4ul of each oligo pool to each tube -> aliquot 100ul of reaction mix into strip tube
Program 95C 30sec -> (95C 3sec -> 55C 30sec-> 72C 20sec) x 16 -> 72C 2min -> 15C hold.
#1-V4 -> 16 cycles #2-V4 -> 16 cycles #3-V4 -> 14 cycles #4-V4 -> 15 cycles (Note: take the average of the qPCR result) File:2012 05 14 Probe-V4-pool.png
- V6 pool for all four sets (12 reaction for each set)
Components | Volume (ul) | 49x rxn mix |
20nM oligo pool | 0.20 | 0.00 |
2x Kapa SYBG qPCR MM | 50.00 | 2,450.00 |
100uM pAP1V61U | 0.40 | 19.60 |
100uM AP2V6 | 0.40 | 19.60 |
2mM aa-dUTP | 3.00 | 147.00 |
H2O | 46.00 | 2,254.00 |
Total volume (ul) | 100.00 | 4,900.00 |
- Aliquot 1197.6 ul in 1.5 mL tube, add 2.4ul of each oligo pool to each tube -> aliquot 100ul of reaction mix into strip tube
Program 95C 30sec -> (95C 3sec -> 55C 30sec-> 72C 20sec) x 15 -> 72C 2min -> 15C hold.
#1-V6 -> 15 cycles #2-V6 -> 14 cycles #3-V6 -> 14 cycles #4-V6 -> 14 cycles (Note: take the average of the qPCR result) File:2012 05 14 Probe-V6-pool.png - Ethanol precipitation. - Pool amplified amplicons (total volume 1,200ul each set) into 15mL tube - Add 4ul of Glycoblue - Add 120ul of 3M NaoAc pH5.5 - Add 3mL of 100% EtOH - Put the tubes at -80C for 1hr - Centrifuge at 3,000rmp for 30min, 4C - Remove spnt. add 600ul of 75% EtOH and transfer the DNA pellet to 1.5 mL tube - Spin at 14,000rmp for 5min - Dry the pellet in the hood and resuspend with 100ul H2O - Re-purified each amplicon with two Qiaquick columns, eluted with 40ul EB => total volume 120ul each. - Measure concentration with Nanodrop.