Rui:LabNotes/SingleCell/2011-1-27
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Positive control (cDNA) for PCR based protocol
Continue with 1.26.11 note
Beads purification
*Pure cDNA w/o primer carry-over *1st round of AMpure beads purification, 20ul --> 36ul beads (1.8*) --> 36ul water *2nd round of AMpure beads purification, 30ul --> 21ul beads (0.7* suggested by Alice) --> 36ul water
Concentration
Nanodrop [ng/ul] Qubit [ug/ml] 1st Hues54 38.67 (2.16; 2.07) 3.76 Hues6-ES 58.78 (2.11; 2.27) 6.05 Hues6-EB 55.25 (2.07; 2.04) 6.54 2nd Hues54 19.72 (2.36; 2.35) 1.29 Hues6-ES 40.1 (2.19; 2.24) 2.44 Hues6-EB 35.92 (2.21; 2.14) 2.64 File:First round beads.jpg
File:Second round beads.jpg
Titration of Exonuclease I
*Goal: high enough to remove primer/unspecific amplification on NTC; low enough to avoid interference to cDNA stability and following steps *Positive control: diluted cDNA w/o primer *Negative control: Rui water *Additional note: ds DNA treatment for single cell???
1. Sample arrangement Template P1 primer SSIII Exo buffer ExoI Water Tube1 water 0.125ul 0.33ul 0.1ul 0.025ul 0.875ul Tube2 water 0.125ul 0.33ul 0.1ul 0.1ul 0.8ul Tube3 water 0.125ul 0.33ul 0.3ul 0.2ul 0.5ul Tube4 water 0.125ul 0.33ul 0.6ul 0.4ul 0 Tube5 0.1pg 6-EB cDNA 0.1ul 0.025ul 0.875ul Tube6 0.1pg 6-EB cDNA 0.1ul 0.1ul 0.8ul Tube7 0.1pg 6-EB cDNA 0.3ul 0.2ul 0.5ul Tube8 0.1pg 6-EB cDNA 0.6ul 0.4ul 0
*In tube 1-4, it should need more Exo I to remove P1 primer since there is no RNA template to consume P1 primer. *In tube 5-8, it should be kept P1 primer free and need less Exo I.
2. Procedure: *Skip 65 heat-up, incubate at 50C for 10 min and 70C for 15min, keep on ice for Exonuclease I treatment step *No modification for following steps after Exonuclease I treatment
Screen for PCR based clons
*Plates: NTC-ES; NTC-EB; 10pg-EB; 500pg-EB *8 single colonies per plate *Takara HS Ex Taq system, 50ul/tube *1ul 10uM primers: P3+P5 *94C, 30sec; 60C, 30sec; 72C, 1min30sec; 35 cycles