IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-7-11

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Contents

PCR Results from 7/10

Here are the results from the 24 PCR reactions we did yesterday. About 2/3 of the reactions used PCC 7942 DNA and Peng's primers for amplifying the KaiABC region. The rest were positive controls using BioBrick inserts and primers.

It appears that the 2 mM Mg+ buffer produced the best results. We're not sure whether an annealing temperature of 55° or 65° produces better yield, since our 65° machine was accidentally misprogrammed.

Reactions 17 and 21 yielded bands in the 3kb region, which is the length of our KaiABC extract.

1. Cy7942, Hotstar Taq, - [Mg+], Old NTPs, 65°C, PCR Machine 1;  2. Cy7942, Vent Taq, 2mM [Mg+], Old NTPs, 65°C, PCR Machine 1;  3. Cy7942, Hotstar Taq, - [Mg+], New NTPs, 65°C, PCR Machine 1;   4. Cy7942, Vent Taq, 2mM  [Mg+], New NTPs, 65°C, PCR Machine 1;   5. Cy7942, Vent Taq, 4mM  [Mg+], New NTPs, 65°C, PCR Machine 1;   6. Cy7942, Vent Taq, 6mM  [Mg+], New NTPs, 65°C, PCR Machine 1;   7. Biobrick, Hotstar Taq, - [Mg+], Old NTPs, 65°C, PCR Machine 1;   8. Biobrick, Vent Taq, 2mM [Mg+], Old NTPs, 65°C, PCR Machine 1;   9. Biobrick, Hotstar Taq, - [Mg+], New NTPs, 65°C, PCR Machine 1;   10. Biobrick, Vent Taq, 2mM  [Mg+], New NTPs, 65°C, PCR Machine 1;   11. Biobrick, Vent Taq, 4mM [Mg+], New NTPs, 65°C, PCR Machine 1;   12. Biobrick, Vent Taq, 6mM [Mg+], New NTPs, 65°C, PCR Machine 1;  The numbers correspond to the labeled lanes, and there is a 1kb+ ladder in the first lane in each gel.
1. Cy7942, Hotstar Taq, - [Mg+], Old NTPs, 65°C, PCR Machine 1;
2. Cy7942, Vent Taq, 2mM [Mg+], Old NTPs, 65°C, PCR Machine 1;
3. Cy7942, Hotstar Taq, - [Mg+], New NTPs, 65°C, PCR Machine 1;
4. Cy7942, Vent Taq, 2mM [Mg+], New NTPs, 65°C, PCR Machine 1;
5. Cy7942, Vent Taq, 4mM [Mg+], New NTPs, 65°C, PCR Machine 1;
6. Cy7942, Vent Taq, 6mM [Mg+], New NTPs, 65°C, PCR Machine 1;
7. Biobrick, Hotstar Taq, - [Mg+], Old NTPs, 65°C, PCR Machine 1;
8. Biobrick, Vent Taq, 2mM [Mg+], Old NTPs, 65°C, PCR Machine 1;
9. Biobrick, Hotstar Taq, - [Mg+], New NTPs, 65°C, PCR Machine 1;
10. Biobrick, Vent Taq, 2mM [Mg+], New NTPs, 65°C, PCR Machine 1;
11. Biobrick, Vent Taq, 4mM [Mg+], New NTPs, 65°C, PCR Machine 1;
12. Biobrick, Vent Taq, 6mM [Mg+], New NTPs, 65°C, PCR Machine 1;
The numbers correspond to the labeled lanes, and there is a 1kb+ ladder in the first lane in each gel.
13. Cy7942, Vent Taq, 2mM [Mg+], New NTPs, 55°C, PCR Machine 1;  14. Cy7942, Vent Taq, 4mM [Mg+], New NTPs, 55°C, PCR Machine 1;  15. Cy7942, Vent Taq, 6mM [Mg+], New NTPs, 55°C, PCR Machine 1;  16. Biobrick, Vent Taq, 2mM [Mg+], New NTPs, 55°C, PCR Machine 1;  17. Cy7942, Vent Taq, 2mM [Mg+], New NTPs, 65°C, PCR Machine 2;  18. Cy7942, Vent Taq, 4mM [Mg+], New NTPs, 65°C, PCR Machine 2;  19. Cy7942, Vent Taq, 6mM [Mg+], New NTPs, 65°C, PCR Machine 2;  20. Biobrick, Vent Taq, 2mM [Mg+], New NTPs, 65°C, PCR Machine 2;  21. Cy7942, Vent Taq, 2mM [Mg+], New NTPs, 55°C, PCR Machine 2;  22. Cy7942, Vent Taq, 4mM [Mg+], New NTPs, 55°C, PCR Machine 2;  23. Cy7942, Vent Taq, 6mM [Mg+], New NTPs, 55°C, PCR Machine 2;  24. Biobrick, Vent Taq, 2mM [Mg+], New NTPs, 55°C, PCR Machine 2;  The numbers correspond to the labeled lanes, and there is a 1kb+ ladder in the first lane in each gel.
13. Cy7942, Vent Taq, 2mM [Mg+], New NTPs, 55°C, PCR Machine 1;
14. Cy7942, Vent Taq, 4mM [Mg+], New NTPs, 55°C, PCR Machine 1;
15. Cy7942, Vent Taq, 6mM [Mg+], New NTPs, 55°C, PCR Machine 1;
16. Biobrick, Vent Taq, 2mM [Mg+], New NTPs, 55°C, PCR Machine 1;
17. Cy7942, Vent Taq, 2mM [Mg+], New NTPs, 65°C, PCR Machine 2;
18. Cy7942, Vent Taq, 4mM [Mg+], New NTPs, 65°C, PCR Machine 2;
19. Cy7942, Vent Taq, 6mM [Mg+], New NTPs, 65°C, PCR Machine 2;
20. Biobrick, Vent Taq, 2mM [Mg+], New NTPs, 65°C, PCR Machine 2;
21. Cy7942, Vent Taq, 2mM [Mg+], New NTPs, 55°C, PCR Machine 2;
22. Cy7942, Vent Taq, 4mM [Mg+], New NTPs, 55°C, PCR Machine 2;
23. Cy7942, Vent Taq, 6mM [Mg+], New NTPs, 55°C, PCR Machine 2;
24. Biobrick, Vent Taq, 2mM [Mg+], New NTPs, 55°C, PCR Machine 2;
The numbers correspond to the labeled lanes, and there is a 1kb+ ladder in the first lane in each gel.

Gel purification

We extracted and purified the 3kb band from reactions 21. According to the nanodrop, we had a DNA concentration of 9.5 ng / µL.

Second gel electrophoresis

We ran a second gel electrophoresis with reactions 17 and 21 as backups in case our transformation failed (see next entry). The results are shown below. We extracted reaction 21 (third lane) and refrigerated it (it hasn't been purified yet).

Second PCR using PCR reactions 17 and 21 (second and third lanes respectively)
Second PCR using PCR reactions 17 and 21 (second and third lanes respectively)


Transformation

We ligated our purified DNA from reaction 21 with TOPO vectors, using Invitrogen's Zero Blunt PCR cloning kit (protocol here). Zero Blunt was used because Vent produces blunt-ended DNA. We then transformed the Topo vector + insert with TOP10 cells using the protocol described here, and incubated overnight, along with a positive and negative control.
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