Rui:LabNotes/SingleCell/2011-1-27

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Positive control (cDNA) for PCR based protocol

Continue with 1.26.11 note

[1]

Beads purification

*Pure cDNA w/o primer carry-over
*1st round of AMpure beads purification, 20ul --> 36ul beads (1.8*) --> 36ul water
File:First round beads.jpg
*2nd round of AMpure beads purification, 30ul --> 21ul beads (0.7* suggested by Alice) --> 36ul water
File:Second round beads.jpg

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

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