Rui:LabNotes/SingleCell/2011-1-28
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Titration Exonuclease I (continued)[edit]
Continued with 1.27.11 lab note[edit]
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
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]