Rui:LabNotes/SingleCell/2011-1-27
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Positive control (cDNA) for PCR based protocol[edit]
Continue with 1.26.11 note[edit]
Beads purification[edit]
- 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[edit]
Nanodrop [ng/ul] V(ul) Total Qubit [ug/ml] Ori.Hues54 414.50 3 1.2ug Hues6-ES 641.13 3 1.8ug Hues6-EB 561.99 3 1.5ug 1st Hues54 38.67 (2.16; 2.07) 36 1.4ug 3.76 Hues6-ES 58.78 (2.11; 2.27) 36 2.1ug 6.05 Hues6-EB 55.25 (2.07; 2.04) 36 1.9ug 6.54 2nd Hues54 19.72 (2.36; 2.35) 36 0.7ug 1.29 Hues6-ES 40.1 (2.19; 2.24) 36 1.4ug 2.44 Hues6-EB 35.92 (2.21; 2.14) 36 1.2ug 2.64
Toughts[edit]
- cDNA synthesis range from 150-?kb, centered around 400bp
- After beads purification, size shifts up and the range is similar as PCR result for 500pg RNA
- samples lost ~50% after 1 round beads purification
Trouble shooting for PCR based protocol[edit]
Continued with 1.26.11 lab note[edit]
Titration of Exonuclease I[edit]
- 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
Sample arrangement[edit]
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.
Procedure[edit]
*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
Result[edit]
Screen for PCR based clons[edit]
- Continue with 1.26.11 lab note [4]
- Screen
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
- Result as in [5]