Noi/NOTES/2012-4-23

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Link to calendar: [[1]]

PCR amplification of circularized DNA captured with probes synthesized by LC Sciences (Library-free protocol), continued[edit]

Plans[edit]

  1. Order two barcoded primers for LC Sciences' library-free protocol, and also sequencing read2 primer
  2. Amplify PGP1-F PGP1-iPS and H1 captured with 4K LC Sciences' probes using library-free protocol in 100ul
  3. Amplify RC-1 library from Baylor for PE sequencing with the new barcoded primer.

1. Order two barcoded primers for LC Sciences' library-free protocol
CA-2-FA.Indx77Sol 5'- CAAGCAGAAGACGGCATACGAGATACCCGTCGGTCTGCCATCCGACGGTAGTGT -3' (Barcode: ACCCGT)
CA-2-FA.Indx78Sol 5'- CAAGCAGAAGACGGCATACGAGATAACACGCGGTCTGCCATCCGACGGTAGTGT -3' (Barcode: AACACG)
CP-2-SeqRead2 5'-CGGTCTGCCATCCGACGGTAGTGT-3'
2. Repeat amplification of PGP1-F PGP1-iPS and H1 captured with LC Sciences' probes using library-free protocol

Components Volume (ul) 4.2 rxn mix
Circularized DNA template 10.00 0.00
10uM CA-2-FA.Indx.Sol 2.00 0.00
10uM CA-2-RA.Sol 2.00 8.40
2x KAPA SYBG MM 50.00 210.00
H2O 36.00 151.20
Total 100.00 420.00
  • aliquot 88 ul, add 2ul CA-2-FA.Indx.Sol primer and 10ul circularized DNA
Samples Index
NTC CA-2-FA.Ind7.Sol
PGP1-F 54C CA-2-FA.Ind7.Sol
PGP1-iPS 54C CA-2-FA.Ind45.Sol
H1 54C CA-2-FA.Ind76.Sol

Program
98°C 30sec -> (98°C 10sec -> 52°C 30sec -> 72°C 30sec) x8 cycles -> (98°C 10sec -> 72°C 30sec) x15 cycles -> 72°C 3 min -> 15°C hold
File:2012 04 22 Library-free LC PGP1f-PGP1iPS-H1.png

2012_04_24[edit]

  • Plan #3: Amplify RC-1 library from Baylor for PE sequencing with the new barcoded primer.
  • Refer to the previous experiment: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2011-12-13
  • Resuspend lyophilized primer from IDT to 100uM by
    • CA-2-FA.Indx77Sol: add 287ul H2O to 28.7nmole
    • CA-2-FA.Indx78Sol: add 298ul H2O to 29.8 nmole
  • Dilute to 10uM total volume 200ul by adding 20ul of 100uM primer to 180ul H2O
  • Samples
    • NTC
    • RC-1 --> barcode: CA-2-FA.Indx77.Sol
Components Volume (ul) 2.2x rxn mix
Pre-amplified DNA Template 2 0
10 uM CA-2-FA.Indx77.Sol 2 4.4
10 uM CA-2-RA.Sol 2 4.4
2X KAPA MM 50 110
H2O 44 96.8
Total 100 220
  • Aliquot 98ul, add 2ul of pre-amplified DNA template

Program
98C 30sec --> (98 C 10 sec --> 54C 30sec --> 72C 30sec) x 4 --> 72 C 3 min
File:2012 04 24 RC-1 Ind77 qPCR.png

  • Purified amplified amplicons with 0.8 vol. AMPure beads and elute with 40ul EB buffer (also include PGP1-F, PGP1-iPS and H1 libraries).
  • Perform PAGE quantification by loading 1ul of purified amplicon in 6% TBE gel.

File:ZhangLab 2 2012-04-24 19hr 57minPQ-labeled.jpg

Samples Conc. (ng/ul) Total yields in 40u (ng)l Volume for 35ng (ul)
PGP1-F 3.92 156.95 8.92
PGP1-iPS 3.04 121.58 11.52
H1 3.90 156.12 8.97
RC-1 0.93 37.38 37.45


  • Perform PAGE-size selection in 5-well 6% TBE gel (2 gels) and resuspend with 20ul H2O

File:ZhangLab 2 2012-04-24 20hr 10min PAGE-ss.jpg

  • Quantify concentration of sequencing library by Qubit dsDNA assay and PAGE quantification (loaded sample 1ul)
    • Qubit dsDNA assay: 1.69ng/ul or 7.9nM (according to the size approx. 350bp. However the concentration will be quantified by Alan again (6.83nM).
    • PAGE quantification: ng/ul or nM

File:ZhangLab 2 2012-04-25 11hr 25min PQ SeqLib.jpg