Rui:LabNotes/SingleCell/2011-1-28: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Zsakura2
>Zsakura2
Line 20: Line 20:
===Thoughts===
===Thoughts===
* ExoI is the key point for the unspecific amplification
* 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.


==Clone screen results (continued)==
==Clone screen results (continued)==

Revision as of 20:14, 24 February 2011

Titration Exonuclease I (continued)

Continued with 1.27.11 lab note

[1]

Results

*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

  • 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.

Clone screen results (continued)

  • Continue with 1.27.11 lab note [2]
  • 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???