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Week 3: May 30-June 3
From 2011.igem.org
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The 6:1 ratio colony that seemed to work based on the gel electrophoresis was grown overnight. | The 6:1 ratio colony that seemed to work based on the gel electrophoresis was grown overnight. | ||
| - | ===Saturday | + | ===Saturday=== |
====Reporters==== | ====Reporters==== | ||
=====PCR Assembly===== | =====PCR Assembly===== | ||
Revision as of 16:39, 7 June 2011
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/μl) | 260/280 |
|---|---|---|
| J33240 | 22.9 | 1.93 |
| R0010 | 137.3 | 1.90 |
We used this information to calculate the concentration (in picomoles/microliter) of our insert and vector DNA. The calculation we used was:
(concentration of DNA)/[(0.66)*(size of part in base pairs)
We wanted 0.25 pM of vector and 0.50 pM of insert, so we calculated how much volume of the insert and vector digests we needed to add for our ligation. These volumes as well as the molar concentrations of our minipreps are summarized in the below table.
| Registry Part | Molar Concentration (pM/μl) | Volume for Ligation (μl) |
|---|---|---|
| J33204 | 0.0119 | 21.0 |
| R0010 | 0.223 | 2.24 |
Restriction Digest
- Insert with XbaI and PstI
J33204
- Note: the following changes were made from the digest protocol:
| dH20: | 39.26 μl |
| J33204: | 2.24 μl |
- Vector with SpeI and PstI
R0010
- Note: the following changes were made from the digest protocol:
| dH20: | 21.0 μl |
| R0010: | 21.0 μl |
Ligation
To get a 4:1 ratio of insert DNA to vector DNA, the following changes were made to the ligation protocol:
| Insert (J33240): | 2 μl |
| Vector (R0010): | 4 μl |
To get a 6:1 ratio of insert DNA to vector DNA, the following changes were made to the ligation protocol:
| Insert (J33204): | 1.5 μl |
| Vector (R0010): | 4.5 μl |
Transformation/Plating
The above ligation was transformed into Escherichia coli cells and plated onto Amp resistant plates.
Friday
Colony PCR
- 4:1 ratio colonies
- 6:1 ratio colonies
- Note: the 4:1 ratio colonies grew a lot more than the 6:1 colonies
Agarose Gel Electrohporesis
- 4:1 colony PCR products
- 6:1 colony PCR products
Results: One of the 6:1 ratio constructs contained the correct amount of base pairs (about 1100). The other constructs did not have more than 500 base pairs. The 6:1 ratio construct will be sequenced to determine its legitimacy.
PCR Assembly
Now that our primers, which were designed last week, arrived, we started constructing the linkers and cleavage sites via PCR. The registry names and our colloquial names (names we refer to these parts as throughout this notebook) are summarized in the following table. We followed the Assembly via PCR protocol for these assemblies.
| Registry Name | Colloquial Name |
|---|---|
| K243004 | small linker |
| K105012 | long linker |
| K316007 | Imperial linker |
| N/A | tev cleavage site |
| N/A | cI cleavage site |
- Note: When purifying the PCR products, DNA wash buffer without ethanol was used, making all of the PCR products unusable.
Culture
The 6:1 ratio colony that seemed to work based on the gel electrophoresis was grown overnight.
Saturday
Reporters
PCR Assembly
The PCR assembly and parts from 6/3/11 were repeated. The correct purification occurred. A gel electrophoresis analysis showed that the PCR assemblies worked.
