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Week 3: May 30-June 3
From 2011.igem.org
Contents |
Monday
Reporters
PCR
J33204 miniprep from 5/26/11
Restriction Digest
- Insert with XbaI and PstI
J33204
- Vector with SpeI and PstI
R0010
Ligation
J33204+R0010 digests
Transformation/Plating
The above ligation was transformed into Escherichia coli cells and plated on Amp resistant plates.
Tuesday
Reporters
Colony PCR
Two colonies from plates from 5/30/11
Agarose Gel Electrophoresis
- J33240 PCR products
- Colony PCR products from above
- Results: The gel showed that the insert (J33240) PCR worked, but the assembly did not work. We came to this conclusion because the constructs on the gel were less than 500 base pairs and our desired construct should have been about 1100 base pairs. Thus, we needed to do assembly for a third time. We started this third assembly with the R0100 miniprep from 5/26/11 and the J33204 PCR products.
Restriction Digest
- Insert with XbaI and PstI
J33204
- Vector with SpeI and PstI
R0010
Ligation
J33204+R0010 digests
Transformation/Plating
The above ligation was transformed into Escherichia coli cells and plated onto Amp resistant plates.
Wednesday
Reporters
Colony PCR
Colonies from plates from 5/31/11 =====Agarose Gel Electrophoresis Colony PCR products from above
- The gel showed that the assembly did not work. The gel had the same results of the gel from last week and the one from 5/31/11. We determined that we need to use a more specific ratio of insert DNA to vector DNA. We will determine this ratio in our next assembly. The next assembly began with transformation of J33204 and R0010 from the registry into Escherichia coli cells.
Thursday
Reporters
Miniprep
- J33204
- R0010
PCR
J33204
Nanodrop
We needed to determine the concentration of DNA in our miniprep products, so we used a nanodrop. Our results are shown in the following table.
| Registry Part | Concentration (ng/ml) | 260/280 |
|---|---|---|
| J33240 | 22.9 | 1.93 |
| R0010 | 137.3 | 1.90 |
