Noi/NOTES/2012-5-14
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File:Example.jpg* [Link to calendar]
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. Probe IDs Conc. (ng/ul) Yields in 120ul (ug) #1-V4 83.70 10.04 #2-V4 125.10 15.01 #3-V4 110.10 13.21 #4-V4 133.80 16.06 #1-V6 122.50 14.70 #2-V6 106.20 12.74 #3-V6 132.20 15.86 #4-V6 101.70 12.20
DpnII digestion
- Note: Since I run out of DpnII and the new order haven't come in yet. I have old tube already expired 07/2011 ~20ul, but still works and the old tube from Dinh (expired 07/09 -> not sure about activity). To test if enzyme still works well then I set up digestion reaction in 50ul and also vary DNA amount between 2.5ug and 4ug to see if 4ul of enzyme can digest 4ug of DNA in 50ul completely or not.
File:ZhangLab 2 2012-05-14 19hr 20min DpnII-digestion-30min.jpg File:ZhangLab 2 2012-05-14 19hr 20min DpnII-digestion-1hr.jpg