Team:Toronto/Notebook-w07-tue
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Tuesday June 28, 2016
Tuesday, 6/28
Members Present:Celine, Karim, Hamed, Kat, Marc
LAB:
Morning:
- Updated transformation results:
- Making buffers for a new batch of RbCl competent cells to be made in the cold room
- Desiging PCR gradient and running PCR on the TetO_LacZ construct:
Manual for Phusion polymerase PCR from thermo:
Thermo Phusion Polymerase Reaction Set Up for Short Linear TetO-LacZ alpha (20uL)
A | B | |
1 | Component | Amount added (uL) |
2 | nuclease free water | |
3 | +MgCl reaction (sample 10) | 11.75 |
4 | +DMSO reaction (samples 11,12) | 12 |
5 | Normal (samples 1-9) | 12.6 |
6 | 10mM dNTPs | 0.4 |
7 | MgCl2 (sample 10 -> +0.2mM) | 0.25 |
8 | Fwd Primer (10uM ->0.5uM) | 1 |
9 | Rev Primer (10uM ->0.5uM) | 1 |
10 | Template DNA (10ng/uL) | 0.5 |
11 | DMSO (sample 11) | 0.6 |
12 | Phusion DNA polymerase | 0.5 |
13 | Final Volume | 20 |
Table2
PCR conditions for Short Linear TetO-LacZ-alpha fragment
A | B | C | D | E | F | G | H | |
1 | Temp | Time | Stage | 20ul Samples run (HF Buffer) | w/ GC Buffer instead of HF buffer | +MgCl | +DMSO | w/ GC Buffer instead of HF buffer AND +DMSO |
2 | 98C | 30s | Initial denaturation | N/A | ||||
3 | 98C | 10s | Denaturation | N/A | ||||
4 | 71.8C | 30sec | Annealing | 1 | ||||
5 | 71.6C | 2 | ||||||
6 | 71.1C | 3 | ||||||
7 | 70.2C | 4 | 9 | 10 | 11 | 12 | ||
8 | 69.1C | x30 | 5 | |||||
9 | 68.2C | 6 | ||||||
10 | 67.6C | 7 | ||||||
11 | 67.3C | 8 | ||||||
12 | 72C | 15s | Extension | N/A | ||||
13 | 72C | 7m | Final Extension | N/A | ||||
14 | 4C | forever | Hold | N/A | ||||
Table1
Gradient PCR for the LacZ fragment. We explored alternate PCR mixes for the calculated optimal annealing temperature for our primers (70.2C). Instead of using the standard HF buffer, we used the GC buffer - both with and without the addition of DMSO. We also explored the addition of DMSO on its own. We also tried optimizing the Mg+ concentration, accounting for the effect of chelators such as EDTA (pressent in TE buffer, which all of our constructs are resuspended in) According to guidelines, we attempted to shift the Mg+ concentration higher by 0.2mM.
Afternoon:
- PCR purification of LacZ gradient PCR samples
- Nanodropping PCR results:
A | B | C | D | |
1 | Sample ID # | 260/280 | 260/230 | concentration (ng/uL) |
2 | 1 | 1.68 | 1.23 | 15.7 |
3 | 2 | 1.68 | 2.09 | 23.1 |
4 | 3 | 1.69 | 1.32 | 33.9 |
5 | 4 | 1.85 | 1.15 | 12.1 |
6 | 5 | 1.75 | 1.14 | 41 |
7 | 6 | 1.66 | 2.28 | 2.3 |
8 | 7 | 1.97 | 1.83 | 23.4 |
9 | 8 | 1.75 | 0.98 | 19 |
10 | 9 | 1.9 | 1.77 | 12.9 |
11 | 10 | 1.68 | 0.96 | 47.2 |
12 | 11 | 2.04 | 2.61 | 11.9 |
13 | 12 | 2.03 | 2.55 | 10.1 |
Table3
- Running a gel for the PCR purified LacZ:
Gel: The gradient PCR temperatures decrease from left to right for lanes 1-8 and 10-13. Lanes 1-8 correspond to samples 1-8. Lane 9 - 3kb ladder. Lanes 10-13 correspond to samples 9-12 (in order). Lane 14 - PCR amplified pSB1C3 backbone from the previous day. Lane 15 - Attempt of PCR amplifyin the Short Linear TetO - LacZ alpha from the previous day.
Issue resolved after consulting Christian: It appears as if our inserts are concatemerizing as we are amplifying linear inserts. What this means is that we are getting fragments with increasing numbers of LacZ adjoined to eachother. Note that the lowest band is at ~400, the next at ~800, followed by ~1600
TO DO:
For the next day:
LAB TEAM:
- Continue with RbCl competent cell protocol (remember to use the cold room)
- If PCR for today is successful, conduct PCR on all the constructs
LAB MANAGERS:
- get Kat her USB! (please and thank you)
- purchase a pair of scissors
- 3kb DNA ladder (can wait until next week; promega 3kb should be fine)
