Noi/NOTES/2012-6-5
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Randomly tagging primer experiment[edit]
Sanger sequencing results of the amplicons amplified with randomly tagging primers (with USER)[edit]
AmpF7AUSol: AATGATACGGCGACCACCGAGAUCUACACAAAAAAACACUCUCAGAUGTUAUCGAGGUCCGAC
AmpF7AUSol: AATGATACGGCGACCACCGAGAUCUACACNNNNNNNCACUCUCAGAUGTUAUCGAGGUCCGAC
Syb_FP5A: AATGATACGGCGACCACCGAG
Syb_RP7: CAAGCAGAAGACGGCATACGAG
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[edit]
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)[edit]
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[edit]
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[edit]
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[edit]
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