Noi/NOTES/2012-6-5: Difference between revisions

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  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)
  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| 400px]]
  [[File:2012_06_05_size-screening-2U.png| 600px]]
    
    
  [[File:2012_06_05_size-screening-5U.png| 400px]]
  [[File:2012_06_05_size-screening-5U.png| 600px]]
    
    
  Note: Loading 5ul of PCR product is overloaded in a small well (26-well comb, 2% SYBR safe gel)
  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 ==

Revision as of 00:57, 6 June 2012

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

Screen more clone 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