Matthew Cai:LabNotes/Probe Production and Capture: Difference between revisions

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==Experimental Plan==
==Experimental Plan==
<!--Comment-->
<!--(1)    Aliquot the seed oligos in the master tube, and perform expansion PCR on one small aliquot (Noi). (3/6)
 
(2)    Perform production PCR followed by nicking enzyme digestion and PAGE size selection (Noi, Matt) (3/7-3/8)
 
(3)    Prepare human gDNA, total RNA, and double-stranded cDNA for capture. gDNA can be from any human cells, such as the Hapmap sample. Total RNA should be the human brain reference RNA (get an aliquot from Rui). cDNA need to be prepared from total RNA through first strand synthesis (NEB E6300S) and second-strand synthesis (NEB E6111S). Please order both kits immediately. (Matt) (3/7-3/8)
 
(4)    Perform padlock capture on the three templates (gDNA, total RNA, ds-cDNA). Note that this probe set has no gap, so you only need Ampligase, but no Stoffel and dNTP. After exo digestion, proceed with PCR. You will need to design PCR primers that can anneal to the common linkers, and contain Illumina adaptors on the 5’ends. The common linker could be too short for forward and reverse primers. In case you can’t design good primers, please look into the barcodes. There are only four different sequences at the end of the barcodes, so you can simply design one common forward primers, and four reverse primers matching the barcodes. (Noi, Matt) (3/11-3/15)
 
(5)    In addition to quantifying the capturing efficiencies, we also want to quantify the abundance of different probes in the initial oligo pool. For this, design two additional primers with Illumina adaptors on the 5’-ends, and eMIP_CA1/eMIP_CA2 on the 3’-ends. Use this primer pair to amplify a small aliquot of the original seed oligos for Illumina sequencing. (Noi, Matt) (3/18-3/22)-->


===Oligo Resuspension===
===Oligo Resuspension===

Revision as of 01:28, 27 February 2013

Experimental Plan

Oligo Resuspension

  • After receiving the probes, resuspend with H2O to the concentration 20 nM (in case it is lyophilized)
  • Perform expansion PCR to amplify the oligoes as the template

Oligo Expansion

Components Volume (ul) Final conc.
20nM LC Sciences Oligoes 10.00 1nM
eMIP_CA1_F (100uM) 0.80 400nM
eMIP_CA1_R (100uM) 0.80 400nM
2x Kapa SYBG MM 100.00 1x
H2O 88.40
Total 200.00

Program
95C 30sec -> (95C 5sec -> 52C 60sec-> 72C 30sec) x 5 -> (95C 5sec -> 60C 30sec-> 72C 30sec) x 10 -> 72C 2min -> 15C hold

  • Primer info.
    • eMIP_CA1_F: TGCCTAGGACCGGATCAACT (TGCCT overhang), Tm of the short one = 53.06C, the long one = 63.28C
    • eMIP_CA1_R: GAGCTTCGGTTCACGCAATG (GAGCT overhang), Tm of the short one = 54.41C, the long one = 62.95C
  • Note:
    • The first five cycles, Tm of each primer was calculated without 5 nt overhang, then the Tm was increased based on the Tm of the full length primer
    • The number of cycles will be monitored
  • Purified with Qiaquick column (2 columns) and elute with EB buffer volume 50ul each --> total volume = 100ul
  • Measure DNA conc. with Nanodrop: 18ng/ul or 295.5nM
  • Dilute 1st round amplicon to 10nM volume 1000ul (mix 33.89ul of 295.5nM 1st round amplicon with 966.11ul H2O)--> for using as the template for the future amplification

Production PCR