Noi/NOTES/2012-6-5

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Randomly tagging primer experiment

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

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


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)

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

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

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

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