Noi/NOTES/2012-6-3
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Randomly tagging primer experiment[edit]
- Continued from: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2012-6-1
- There are a lot of colonies on the the plates, this suggests the transformation worked very well (no negative control was performed). On the plates plated with 50ul transformed E. coli showed single colony that can be picked up for PCR better than the plates plated with 150ul which have very close colonies and difficult to pick up individual colonies
Colony PCR[edit]
- Lysed the cells in 10 ul H2O by using 10ul pipette tip to pick up the white single colony and pipette up and down in the 96-well plate containing 10ul H2O - heated at 98C for 10 min - vortexed vigorously - put the tube on ice box and spin down the cell lysis
Components | 1 rxn | 84 rxn mix |
Cell lysis | 2.00 | 0.00 |
10uM Syb_FP5A | 1.00 | 84.00 |
10uM Syb_RP7 | 1.00 | 84.00 |
2X Taq MM | 25.00 | 2,100.00 |
H20 | 21.00 | 1,764.00 |
Total | 50.00 | 4,200.00 |
- Aliquot 48ul, add 2ul of cell lysis DNA template
Program 96C 3min -> (95C 30s -> 58C 45s -> 72C 45s)x36 cycles --> 72C 5min --> hold at 15C Note: Always get evaporation problem of the samples in the edge of the 96-well plate (long time amplification ~3hr) even sealing very well 2012_06_04 - Analyzed in 2% E-gel by loading PCR product 10ul File:ZhangLab 2 2012-06-04 10hr 44min EGel1.jpg - Select 20 positive clones from each USER volume (later) - Purified with 1vol. AmPure beads and eluted with 30ul EB buffer - Measured DNA conc. by Nanodrop
DNA preparation for Sanger sequencing at GENEWIZ[edit]
- I will send 10 clones from 1U first, if the sequencing give the right sequences, I would send more for the rest (total 22 clones per USER volume) - In total 15ul, put 2.5 ul of 10uM Syb_RP7 primer, 50ng of bead purified DNA, and adjusted volume to 15 ul with H2O Sample ID Conc. (ng/ul) ~ Volume for 50ng (ul) 1U-1 38.9 1.25 1U-2 42.8 1.25 1U-3 45.0 1.25 1U-4 44.5 1.25 1U-5 48.0 1.25 1U-6 42.9 1.25 1U-7 40.0 1.25 1U-8 39.0 1.25 1U-9 44.0 1.25 1U-10 39.9 1.25 H2O --> 11.25ul, 10uM Syb_RP-7 --> 2.50ul Tracking number: 10-196380326: under Dinh's account PO#: 90443171: generated by Rui (Total 60, 2012_06_04 send 10 samples => 50 reactions left)