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= Randomly tagging primer experiment = == Sanger sequencing results of the amplicons amplified with randomly tagging primers (with USER) == * Continued from: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2012-6-5 == 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 == {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Sample IDs''' | align="center" style="background:#f0f0f0;"|'''Surrounding sequences<br>contain AGAGTG (7A or 7N) GTG''' | align="center" style="background:#f0f0f0;"|'''7A, AAAAAAA''' | align="center" style="background:#f0f0f0;"|'''Note:''' | align="center" style="background:#f0f0f0;"|'''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| 450px]] [[File:2U-2.png| 450px]] [[File:2U-3.png| 450px]] [[File:2U-4.png| 450px]] [[File:2U-5.png| 450px]] [[File:2U-6.png| 450px]] [[File:2U-7.png| 450px]] [[File:2U-8.png| 450px]] [[File:2U-9.png| 450px]] [[File:2U-10.png| 450px]] [[File:2U-13.png| 450px]] [[File:2U-14.png| 450px]] [[File:2U-15.png| 450px]] [[File:2U-16.png| 450px]] == Chromatograms of the clones need to be re-sequenced == [[File:2U-11.png| 450px]] [[File:2U-12.png| 450px]] == Re-sequencing of clone 2U-11 and 2U-12 == * 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| 450px]] [[File:2U-12_R.jpg| 450px]]<br> '''Summary''' Total 16 clones of 2U (2ul USER), all 16 clones have no the fragment amplified with AmpFNU.Sol == 5ul USER/5U == {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Sample IDs''' | align="center" style="background:#f0f0f0;"|'''Surrounding sequences<br>contain AGAGTG (7A or 7N) GTG''' | align="center" style="background:#f0f0f0;"|'''7A, AAAAAAA''' | align="center" style="background:#f0f0f0;"|'''Note:''' | align="center" style="background:#f0f0f0;"|'''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 == [[File:5U-1.png| 450px]] [[File:5U-3.png| 450px]] [[File:5U-4.png| 450px]] [[File:5U-7.png| 450px]] [[File:5U-8.png| 450px]] [[File:5U-9.png| 450px]] [[File:5U-10.png| 450px]] [[File:5U-11.png| 450px]] [[File:5U-14.png| 450px]] [[File:5U-15.png| 450px]] [[File:5U-16.png| 450px]] == Chromatograms of the clones need to be re-sequenced == [[File:5U-2.png| 450px]] [[File:5U-5.png| 450px]] [[File:5U-6.png| 450px]] [[File:5U-12.png| 450px]] [[File:5U-13.png| 450px]] == Re-sequencing of clone 5U-2, 5U-5, 5U-6, 5U-12 and 5U-13 == * 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| 450px]] [[File:5U-5_R.png| 450px]] [[File:5U-6_R.png| 450px]] [[File:5U-12_R.png| 450px]] [[File:5U-13_R.png| 450px]] == 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) [[File:2012_06_06_Size-screening_1U-1-12.png| 400px]] [[File:2012_06_06_Size-screening_1U-12-24.png| 400px]] - 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) * Continued on: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2012-6-7
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