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]

[1]

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]

[2]

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]

[3]

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]