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= Randomly tagging primer experiment = * Continued from: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2012-6-3 == Sanger sequencing results of the amplicons amplified with randomly tagging primers (with USER)== '''AmpF7AUSol:''' AATGATACGGCGACCACCGAGAUCUACAC'''AAAAAAA'''CACUCUCAGAUGTUAUCGAGGUCCGAC<br> '''AmpF7AUSol:''' AATGATACGGCGACCACCGAGAUCUACAC'''NNNNNNN'''CACUCUCAGAUGTUAUCGAGGUCCGAC<br> '''Syb_FP5A:''' AATGATACGGCGACCACCGAG<br> '''Syb_RP7:''' CAAGCAGAAGACGGCATACGAG<br> '''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 == <br> [[File:1U-1.png| 450px]] [[File:1U-2.png| 450px]] [[File:1U-3.png| 450px]] [[File:1U-4.png| 450px]] [[File:1U-5.png| 450px]] [[File:1U-6.png| 450px]] [[File:1U-7.png| 450px]] [[File:1U-8.png| 450px]] [[File:1U-9.png| 450px]] [[File:1U-10.png| 450px]] * 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| 450px]] [[File:2012_06_04_1U-8_R.png| 450px]] * 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| 600px]] [[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) - 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 == {| {{table}} border = 1 style="text-align: center;" | align="center" style="background:#f0f0f0;"|'''Sample IDs''' | align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''~ Volume for 50ng (ul)''' | align="center" style="background:#f0f0f0;"|'''H2O (ul)''' | align="center" style="background:#f0f0f0;"|'''10uM Syb_RP7 (ul)''' | align="center" style="background:#f0f0f0;"|'''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 == {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1 rxn''' | align="center" style="background:#f0f0f0;"|'''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 * Continued on: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2012-6-6
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