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| | [[File:2012_06_05_size-screening-2U.png| 600px]] | ||
[[File:2012_06_05_size-screening-5U.png| | [[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