Pooled Colony Screening Process
If no insert of the correct size was found in the five colony direct colony screen, a pooled colony screen was used for further analysis.
1. Cells from five colonies were picked using a sterile toothpick and placed into a single tube. This was repeated to give a total of 25 colonies in 5 tubes.
2. Mix PCR cocktail dispense into 96 tubes and complete PCR.
Cocktail 1X 105X
10% sterile glycerol 7.5 µl 787.5 µl
dNTPs (2 mM) 1.875 µl 196.875à 196.9µl
MgCl2 (25 mM) 0.5 µl 52.5µl
10X Taq Polymerase Buffer 1.25 µl 131.25 µl
DMSO 0.625 µl 65.625à 65.6 µl
Taq Polymerase (5 Unit/µl) 0.25 µl 26.25 µl
AttL1 (100 pmole/µl) 0.25 µl 26.25 µl
AttL2 (100 pmole/µl) 0.25 µl 26.25 µl
Templateà sufficient smear of cells from each colony for visible evidence in tube
VT=12.5 µl
Note that the total volume is 12.5 µl. Because the bacterial smear adds nominal volume to the reaction, the entire 12.5 µl is added to each tube.
3. Run the following program on a MJ Research PTC-200 DNA Engine (M J Research, Inc., San Francisco, CA) or similar machine. Expected Run time: ~5 hrs 30 min
1. 95ºC-3 min
2. 94ºC-2 min
3. 94ºC-1 min
4. 67ºC-1 min
5. 68ºC-6 min
6. 94ºC-1 min
7. 66ºC-1 min
8. 68ºC-6 min
9. 94ºC-1 min
10. 65ºC-1 min
11. 68ºC-6 min
12. 94ºC-1 min
13. 64ºC-1 min
14. 68ºC-6 min
15. 94ºC-1 min
16. 63ºC-1 min
17. 68ºC-6 min
18. 94ºC-1 min
19. 62ºC-1 min
20. 68ºC-6 min
21. 94ºC-1 min
22. 61ºC-1 min
23. 68ºC-6 min
24. 94ºC-1 min
25. 60ºC-1 min
26. 68ºC-6 min
27. go to step 24, 32X
28. 68ºC-6 min
29. 4ºC-forever
30. end
When large ORFs (>1500 bp) were to be screened, an extension time of 8 minutes was used.
4. If a well on the gel contained a PCR fragment of the predicted size, the corresponding set of 5 colonies was screened by the Direct Colony Screen Protocol to identify colonies that were carrying a correct plasmid.
5. If the wells on the gel scored negative, the attempt to clone the ORF was reinitiated.