Noi/NOTES/2012-6-6

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Randomly tagging primer experiment[edit]

Sanger sequencing results of the amplicons amplified with randomly tagging primers (with USER)[edit]

Tagging_validation_2012_06_05[edit]

  • 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[edit]

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[edit]

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[edit]

File:2U-11.png File:2U-12.png

Re-sequencing of clone 2U-11 and 2U-12[edit]

  • Note: The sequencing of these two clones were early terminated and the peaks at this region were not very high However, the sequences showed the 7T stretch and the surrounding are most likely correct. This is good enough to conclude that these two clone contain the DNA amplified with AmpFAU.Sol not AmpFNU.Sol

File:2U-11 R.jpg File:2U-12 R.jpg

Summary
Total 16 clones of 2U (2ul USER), all 16 clones have no the fragment amplified with AmpFNU.Sol

5ul USER/5U[edit]

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[edit]

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[edit]

File:5U-2.png File:5U-5.png File:5U-6.png File:5U-12.png File:5U-13.png

Re-sequencing of clone 5U-2, 5U-5, 5U-6, 5U-12 and 5U-13[edit]

  • Note: Re-sequencing didn't give a better result. This is due to the quality of sequencing. I will discuss with Athurva and Dinh to see how we are going to conclude about the sequencing of these clones. Anyway, there are poly T sequences on the expected regions, but only one nucleotide difference or missing of one base.

File:5U-2 R.png File:5U-5 R.png File:5U-6 R.png File:5U-12 R.png File:5U-13 R.png

Screen more clones for Sanger sequencing[edit]

- Perform size screen of the PCR products from 2012_06_05 in 2% agarose gel, load sample 4ul each well (24 clones from 1U)
  
File:2012 06 06 Size-screening 1U-1-12.png   File:2012 06 06 Size-screening 1U-12-24.png  
   
- 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 (Tagging_Validation_2012_06_06), 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)