Rui:LabNotes/SingleCell/2011-1-28

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Titration Exonuclease I (continued)[edit]

Continued with 1.27.11 lab note[edit]

[1]

Results[edit]

*Each samples (tube1 to 8) have 4 aliquots for PCR, 40ul/aliquotes
*4ul PCR products from one aliquot per sample was checked by 6% TBE-PAGE
*30min running time 
Sample arrangement 
        Template         P1 primer   SSIII    Exo buffer    ExoI       Final
Tube1   water            0.125ul     0.33ul   1* in 1ul     0.5U       0.16* in 6ul
Tube2   water            0.125ul     0.33ul   1* in 1ul     2U         0.16* in 6ul
Tube3   water            0.125ul     0.33ul   3* in 1ul     4U         0.5* in 6ul
Tube4   water            0.125ul     0.33ul   6* in 1ul     8U         1* in 6ul
Tube5   0.1pg 6-EB cDNA                       1* in 1ul     0.5U       0.16* in 6ul       
Tube6   0.1pg 6-EB cDNA                       1* in 1ul     2U         0.16* in 6ul
Tube7   0.1pg 6-EB cDNA                       3* in 1ul     4U         0.5* in 6ul
Tube8   0.1pg 6-EB cDNA                       6* in 1ul     8U         1* in 6ul

File:Exo I.jpg

Thoughts[edit]

  • ExoI is the key point for the unspecific amplification
  • No amplification for cDNA positive control could due to the temperature changes during the procedure by which cDNA was degraded by ExoI.
  • 500pg total RNA can still be used as the positive control, which works well under the original protocol. If extra Exonuclease doesn't have bad effect on 500pg, it shouldn't have effect on low RNA amount.
  • Continued with [2]

Clone screen results (continued)[edit]

  • Continue with 1.27.11 lab note [3]
  • Gel check
Upper part: NTC-ES; NTC-EB
Lower part: 10pg-EB; 500pg-EB
5ul out of 50ul PCR products
File:Clone screen.jpg
  • Thoughts??? and trouble shooting as in [4]