Noi/NOTES/2012-6-6

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Revision as of 01:13, 7 June 2012 by >Noi
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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

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

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