Noi/NOTES/2012-6-6
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Randomly tagging primer experiment
Sanger sequencing results of the amplicons amplified with randomly tagging primers (with USER)
Tagging_validation_2012_06_05
- Note:
- All samples showing ":Spectral Pull-up" are free-relaoded
- From previous result of re-sequencing of the samples showing "Homopolymeric or Repetitive Region", the results were pretty much the same because of the presence of poly N region upstream of the 7T sequences that causes the non-specific signal after that region. Therefore I will not repeat sequencing for those clones, but the sequences are good enough for validation
2ul USER/2U
Sample IDs | Surrounding sequences contain AGAGTG (7A or 7N) GTG |
7A, AAAAAAA | Note: | Pass validation |
2U-1 | yes | yes | yes | |
2U-2 | yes | yes | yes | |
2U-3 | yes | yes | yes | |
2U-4 | seem to,yes | most likely yes | :Homopolymeric or Repetitive Region | yes |
2U-5 | seem to,yes | yes | :Homopolymeric or Repetitive Region | yes |
2U-6 | yes | yes | :Spectral Pull-up, free-reload | yes |
2U-7 | most likely yes | yes | :Homopolymeric or Repetitive Region | yes |
2U-8 | yes | yes | yes | |
2U-9 | yes | yes | yes | |
2U-10 | yes | yes | :Spectral Pull-up, free-reload | yes |
2U-11 | not clear | yes, peak not clear | :Spectral Pull-up, free-reload | no: need to confirm by reloading |
2U-12 | not clear | yes, peak not clear | :Spectral Pull-up, free-reload | no: need to confirm by reloading |
2U-13 | yes | yes | :Spectral Pull-up, free-reload | yes |
2U-14 | yes | yes | :Spectral Pull-up, free-reload | yes |
2U-15 | seem to,yes | most likely yes | :Homopolymeric or Repetitive Region | yes |
2U-16 | yes | yes | :Spectral Pull-up, free-reload | yes |
Summary - Total 16 clones - 14 clones correct - 2 clones need to be confirmed by re-sequencing
Chromatograms of the clones pass validation
File:2U-1.png File:2U-2.png File:2U-3.png File:2U-4.png File:2U-5.png File:2U-6.png File:2U-7.png File:2U-8.png File:2U-9.png File:2U-10.png File:2U-13.png File:2U-14.png File:2U-15.png File:2U-16.png
Chromatograms of the clones need to be re-sequenced
File:2U-11.png File:2U-12.png Re-sequencing of clone 2U-11 and 2U-12
- Note: The sequencing of these two clones were early terminated and the peaks at this region were not very clear. However, the sequences showed the 7T stretch and the surrounding are most likely correct. This suggests all 16 clones of 2U all contain 7T not 7N
File:2U-11 R.jpg File:2U-12 R.jpg
5ul USER/5U
Sample IDs | Surrounding sequences contain AGAGTG (7A or 7N) GTG |
7A, AAAAAAA | Note: | Pass validation |
5U-1 | seem to,yes | most likely yes | :Homopolymeric or Repetitive Region | yes |
5U-2 | yes | 6T in stead of 7T | * repeat to confirm 7T | |
5U-3 | seem to,yes | most likely yes | :Homopolymeric or Repetitive Region | yes |
5U-4 | yes | yes | :poor quality | yes |
5U-5 | yes | TCTTTTT | * repeat to confirm | |
5U-6 | unclear | seem to, | :poor quality | ? * repeat to confirm |
5U-7 | yes | yes | yes | |
5U-8 | seem to,yes | yes | :Homopolymeric or Repetitive Region | yes |
5U-9 | yes | yes | :Spectral Pull-up, free-reload | yes |
5U-10 | yes | yes | yes | |
5U-11 | seem to,yes | most likely yes, | :Homopolymeric or Repetitive Region | yes |
5U-12 | not clear | seem to by sequences but not the peak | :Spectral Pull-up, free-reload | no: need to confirm by reloading |
5U-13 | yes | TTTTCTT | :Spectral Pull-up, free-reload | * repeat to confirm 7T |
5U-14 | yes | yes | :Spectral Pull-up, free-reload | yes |
5U-15 | yes | yes | yes | |
5U-16 | seem to, yes | yes | :Homopolymeric or Repetitive Region | yes |
Summary - Total 16 clones - 11 clones correct - 5 clones were repeated for confirmation
Chromatograms of the clones pass validation
File:5U-1.png File:5U-3.png File:5U-4.png File:5U-7.png File:5U-8.png File:5U-9.png File:5U-10.png File:5U-11.png File:5U-14.png File:5U-15.png File:5U-16.png
Chromatograms of the clones need to be re-sequenced
File:5U-2.png File:5U-5.png File:5U-6.png File:5U-12.png File:5U-13.png
Screen more clones for Sanger sequencing
- Perform size screen of the PCR products from 2012_06_05 in 2% agarose gel, load sample 4ul each well (24 clones from 1U, 16 clones from each 2U or 5U) - Purified 30ul of PCR products with 1vol. AmPure beads and eluted with 30ul EB buffer - Measured DNA conc. by Nanodrop - I send more 18 clones of 1U (1U-11 - 1U-28) for Sanger sequencing Tracking number: 10-196614018, under Dinh's account PO#: 90443171: generated by Rui (Total 60, 2012_06_04 send 10 samples, 2012_06_05 send 32 samples , 2012_06_06 send 18 samples => 0 reactions left)