Noi/NOTES/2012-6-6
Jump to navigation
Jump to search
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
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)