Marianne and Vicki
Observations of plates after transformation:
Table 1. Observations of plates
|Plate/Sample||Number of colonies|
|2T||TMTC (too many to count)|
|condensed, white, some bigger than others|
See 'Colony PCR protocol' in 'iGEM Common Protocols". It is written below for easy access:
- PCR tubes
- BioBrick PCR primers (G1004, G1005)
- Taq polymerase
- Buffer, Mg2+, dNTPs
- Agar plate for indexing
- Make master mix of primers and other PCR components (details to be worked out when we get the supplies)
- Add Taq polymerase.
- Aliquot 10uL per PCR tube.
- Touch toothpick/pipet tip/loop to colony, then index plate, then swirl around in PCR tube.
- Run PCR: Knight lab uses this protocol, although when I did cPCR I only did about 30 cycles:
1. 95°C for 15 mins
2. 94°C for 30 secs
3. 56°C for 30 secs
4. 68°C for 1 min per kb of expected product
* I typically round up for this step. i.e. For a 3.6kb construct, I used a 4 min elongation time.
It seems to help to be a bit generous with the elongation time.
5. Repeat 2-4 39 times.
6. 68°C for 20 mins
7. 4°C forever
- Verify PCR products on an agarose gel.
Making the master mix
- Will pick 20 colonies for 1 tube for each plate, for a total of 8 PCR tubes
*1 PCR tube x 8 plates + 2 extra = 10 samples
Table 2. Master Mix for colony PCR
|REAGENT||1 RXN VOLUME (uL)||MASTER MIX (uL)|
|10x rxn buffer||2.5||x10||25|
|10uM FW primer: G1004||2||x10||20|
|10uM RE primer: G1005||2||x10||20|
Table 3. PCR Tubes
|From Plate (Transformants)||To PCR Tubes|
|W (H2O control)|
*Note: For Plate 11Ta, 20th colony is on the 21st square. For 6Ta, 1st colony is on the 2nd square and the 2nd colony is on the 1st sqaure
- Aliquot 25uL to PCR tubes
- Annealing temperature: 63degree C
- Elongation time: 80s
- PCR Cycle:
- 95C @ 2min
- Cycle 30x
- 95C @ 30s
- 63C @ 10s
- 72C @ 80s
- 72C @ 10min
- 10C @ Hold
Start PCR: 1428
End PCR: 1600
Removed and put in 4C fridge. Will gel verify tomorrow.
General Lab Duties
- Autoclaved micropipet tips (10uL and 1000uL)
- Autoclaved LB Agar x2 bottles
Vicki Ma 23:34, 28 June 2010 (EDT)
Eric F. and Jason Gao
Get an OD growth curve for S. Aureus strain RN4220. Growth curve will be used to eliminate the need to measure optical density every time we electroporate.
Therefore we need to follow the electroporation protocol or the data will mean nothing.
- Prepare overnight culture grown in B2 broth with constant aeration at 37 degrees C
- Dilute 1/25 into 25mL of fresh B2 broth in 300mL flask
- Grow with constant aeration at 37 degrees C until reading OD 600 of about 0.4
...other steps not shown
Make B2: Recipe
- 1.0% Casein Hydrolysate
- 2.5% Yeast extract
- 0.1% K2HPO4
- 0.5% Glucose
- 2.5% NaCl
- Adjust pH to 7.5
Decided to make a 400mL batch so...
- 4g Casein Hydrolysate
- 10g Yeast extract
- 0.4g K2HPO4
- 2g glucose
- 10g Nacl
Once everything was mixed the pH was 7.11
To balance the pH we added 2.8mL of 0.3M NaOH, 2 pellets of NaOH, added 1.8mL 1M HCl, the final pH was 7.47
It was made aseptically (without a flame) so the B2 broth was not autoclaved.
Overnight Culture in B2 Broth
- Using 2 14mL round bottom test tubes
- Filled each with 5mL B2 Broth and added a pipette tip to each
- One pipette tip was used to scrape the RN4220 glycerol stock (before being added to the broth)
- both test tubes were placed in the shaky incubator at 1815 at 90 RPM at 37 degrees C
- Must be removed by 1215 June 29th