Noi/NOTES/2012-6-5
Jump to navigation
Jump to search
Randomly tagging primer experiment
Sanger sequencing results of the amplicons amplified with randomly tagging primers (with USER)
Check Sanger's sequencing result roughly before sending more clones
- Primimg with Syb_RP-7: read reverse complementary of the AmpFA/NU.Sol strand
qs (quality score) Sequence correct 7nt=AAAAAAA Note contain AGAGTG(7A or7N)GTG 1U-1 29 seem to (overlapping peaks) yes, :Homopolymeric or Repetitive Region, **request for free repeat 1U-2 29 yes yes, :Homopolymeric or Repetitive Region 1U-3 33 yes yes, chromatogram very clear even the peak very low 1U-4 32 yes yes, 1U-5 42 yes yes, 1U-6 43 yes yes, 1U-7 42 yes yes, chromatogram very clear even the peak very low 1U-8 14 seem to (overlapping peaks) yes Non-specific, **request for free repeat 1U-9 31 yes yes, 1U-10 43 no no, This clone has a shift band higher than other positive clones on E-gel will look closer to the sequences (seem to be neither AmpFNU.Sol nor AmpFAU.Sol)
1ul USER/1U
File:1U-1.png File:1U-2.png File:1U-3.png File:1U-4.png File:1U-5.png File:1U-6.png File:1U-7.png File:1U-8.png File:1U-9.png File:1U-10.png
- Summary: From 10 clones sequenced by Sanger sequencing
- 7 clones are clearly correct and all contain 7T
- 2 clones clearly showed 7T but show overlapping of the peaks surrounding 7T sequences --> request for free repeat
- 1 clone showed unrelated sequences of the clone amplified by AmpFNU.Sol or AmpFAU.Sol
Repeat sequencing of clone 1U-1 (1U-1_R) and 1U-8 (1U-8_R)
File:2012 06 04 1U-1 R.png File:2012 06 04 1U-8 R.png
- From the chromatograms of the two clones re-sequenced, the results are pretty much the same. So in the future, I will not do re-sequencing of the samples showing Homopolymeric or Repetitive Region comment after sequencing. However, I could see that the sequences were correct since there are the poly T (TTTTTTT, 7T) and the peaks of surrounding sequences AGAGTG (TTTTTTT, 7T)GTG exist, but just the peaks that overlap.
- Summary of 1ul USER (1U) 10 clones
- 9 clones showed polyT (TTTTTTT, 7T) sequences
- 1 clone showed sequences not amplified by either AmpFNU.Sol or AmpFAU.Sol
- I will mainly focus on 1ul USER validation rather than 2 or 5ul USER for the moment.
- Additional Sanger sequencing validation of 18 clones of 1U (1ul USER): http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2012-6-7
Screen more clones for Sanger sequencing
Perform size screen of the PCR products from 2012_06_03 in 2% agarose gel, load sample 5ul each well (24 clones from each 2U or 5U) File:2012 06 05 size-screening-2U.png File:2012 06 05 size-screening-5U.png Note: Loading 5ul of PCR product is overloaded in a small well (26-well comb, 2% SYBR safe gel) - Purified 35ul PCR products with 1vol. AmPure beads and eluted with 30ul EB buffer - Measured DNA conc. by Nanodrop
DNA preparation for Sanger sequencing at GENEWIZ
Sample IDs | Conc. (ng/ul) | ~ Volume for 50ng (ul) | H2O (ul) | 10uM Syb_RP7 (ul) | Total volume (ul) |
2U-1 | 41.30 | 1.25 | 11.25 | 2.50 | 15.00 |
2U-2 | 41.30 | 1.25 | 11.25 | 2.50 | 15.00 |
2U-3 | 28.10 | 2.00 | 10.50 | 2.50 | 15.00 |
2U-4 | 39.50 | 1.25 | 11.25 | 2.50 | 15.00 |
2U-5 | 47.40 | 1.25 | 11.25 | 2.50 | 15.00 |
2U-6 | 26.30 | 2.00 | 10.50 | 2.50 | 15.00 |
2U-7 | 19.10 | 2.50 | 10.00 | 2.50 | 15.00 |
2U-8 | 16.20 | 3.00 | 9.50 | 2.50 | 15.00 |
2U-9 | 39.90 | 1.25 | 11.25 | 2.50 | 15.00 |
2U-10 | 42.20 | 1.25 | 11.25 | 2.50 | 15.00 |
2U-11 | 35.70 | 1.50 | 11.00 | 2.50 | 15.00 |
2U-12 | 32.20 | 1.50 | 11.00 | 2.50 | 15.00 |
2U-13 | 36.20 | 1.50 | 11.00 | 2.50 | 15.00 |
2U-14 | 45.80 | 1.25 | 11.25 | 2.50 | 15.00 |
2U-15 | 33.50 | 1.50 | 11.00 | 2.50 | 15.00 |
2U-16 | 35.20 | 1.50 | 11.00 | 2.50 | 15.00 |
5U-1 | 33.50 | 1.50 | 11.00 | 2.50 | 15.00 |
5U-2 | 26.00 | 2.00 | 10.50 | 2.50 | 15.00 |
5U-3 | 22.70 | 2.00 | 10.50 | 2.50 | 15.00 |
5U-4 | 26.10 | 2.00 | 10.50 | 2.50 | 15.00 |
5U-5 | 42.00 | 1.25 | 11.25 | 2.50 | 15.00 |
5U-6 | 41.00 | 1.25 | 11.25 | 2.50 | 15.00 |
5U-7 | 34.90 | 1.25 | 11.25 | 2.50 | 15.00 |
5U-8 | 38.10 | 1.50 | 11.00 | 2.50 | 15.00 |
5U-9 | 46.30 | 1.25 | 11.25 | 2.50 | 15.00 |
5U-10 | 41.80 | 1.25 | 11.25 | 2.50 | 15.00 |
5U-11 | 41.20 | 1.25 | 11.25 | 2.50 | 15.00 |
5U-12 | 30.20 | 1.50 | 11.00 | 2.50 | 15.00 |
5U-13 | 27.50 | 2.00 | 10.50 | 2.50 | 15.00 |
5U-14 | 35.70 | 1.50 | 11.00 | 2.50 | 15.00 |
5U-15 | 38.00 | 1.25 | 11.25 | 2.50 | 15.00 |
5U-16 | 33.30 | 1.50 | 11.00 | 2.50 | 15.00 |
- Aliquot 48ul, add 2ul of cell lysis DNA template
Tracking number: 10-196512843 ( Tagging_validation_2012_06_05): under Dinh's account PO#: 90443171: generated by Rui (Total 60, 2012_06_04 send 10 samples, 2012_06_05 send 32 samples => 18 reactions left)
PCR
Components | 1 rxn | 58 rxn mix |
Cell lysis | 2.00 | 0.00 |
10uM Syb_FP5A | 1.00 | 58.00 |
10uM Syb_RP7 | 1.00 | 58.00 |
2X Taq MM | 25.00 | 1,450.00 |
H20 | 21.00 | 1,218.00 |
Total | 50.00 | 2,784.00 |
Program 96C 3min -> (95C 30s -> 58C 45s -> 72C 45s)x36 cycles --> 72C 5min --> hold at 15C