Noi/NOTES/2012-4-23
Link to calendar: [[1]]
PCR amplification of circularized DNA captured with probes synthesized by LC Sciences (Library-free protocol), continued[edit]
Plans[edit]
- Order two barcoded primers for LC Sciences' library-free protocol, and also sequencing read2 primer
- Amplify PGP1-F PGP1-iPS and H1 captured with 4K LC Sciences' probes using library-free protocol in 100ul
- 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
- The sequencing libraries: NP-BSPP-LC_PGP1FiRC1-Apr24 were sequenced on 120426_HL118 run allocated on lane #5
- Read1: CP-2-SeqRead1.x
- Sample index read: CP-2-IndSeq.x
- Read2: CP-2-SeqRead2
- Summary of mapping data:http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2012-5-7#Mapping_to_hg19_.28on_Triton_cluster.29