02/09/2016
Examining pLas + LasR Ligation Plates
Jiaqi performed ligations with pLas and LasR, and after overnight incubation at 37°C, the plates were as shown in the picture below:
Figure 1 - pLas + LasR Plates
:  The plates exhibited the following distribution of colonies:
- pLas 1 (insert) / 2 (backbone) - Many colonies
- pLas 2/2 - ~15 colonies
- Both of the plates above had a similar number of colonies to the LasR Bb gel 2 plate, which indicates that most of the colonies grown only contained the backbone re-annealed to itself, instead of the backbone with the insert
- pLas 1/1 - ~10 colonies
- pLas 2/1 - 4 colonies
- These two plates above also showed the same pattern of colonization as the pLas plates with Bb 2
- LasR Bb gel 1 - 4 colonies
- LasR Bb gel 2 - Many colonies
- Negative control - 1 colony
- Despite this single colony, it was determined that the other plates were not affected by contamination, as they had a substantial amount of colonies grow on them
Generally speaking, there were many satellite colonies that grew around the larger, major colonies.
Preparing gBlock Positive Control for Sequencing
The general mixture for sequencing consists of...
- 1.6 µL of 10 µM primer
- 500 ng of DNA
- Up to 10 µL of water
The mixtures containing the positive control were prepared as shown in the table below:
Table 1 - gBlock Sequencing Mixtures
| Reagent
|
Sequencing Mixture
|
| 1
|
2
|
3
|
4
|
5
|
| Primer (µL) |
1.6 |
1.6 |
1.6 |
1.6 |
1.6
|
| DNA (µL) |
3.8 |
4.3 |
4.4 |
5.7 |
5.1
|
| Water (µL) |
4.6 |
4.1 |
4.0 |
2.7 |
3.3
|
| Total (µL) |
10.0 |
10.0 |
10.0 |
10.0 |
10.0
|
The sequencing mixture numbers given in the table above correspond to the following plasmids:
- 1 = gB015 1
- 2 = gB015 2
- 3 = gB015 3
- 4 = gB015 gel 1
- 5 = gB015 gel 2
| 
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