IGEM:UBC/2009/Notebook/UBC iGEM 2010/2010/06/28

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dspB Track

Marianne and Vicki

Colony PCR

Observations of plates after transformation:

Table 1. Observations of plates
Plate/SampleNumber of colonies
2TTMTC (too many to count)
4TTMTC
5TTMTC
6TTMTC
8TTMTC
10TTMTC
11TTMTC
12TTMTC
condensed, white, some bigger than others

See 'Colony PCR protocol' in 'iGEM Common Protocols". It is written below for easy access:
Supplies Needed:

  • PCR tubes
  • BioBrick PCR primers (G1004, G1005)
  • Taq polymerase
  • Buffer, Mg2+, dNTPs
  • Agar plate for indexing

Steps:

  1. Make master mix of primers and other PCR components (details to be worked out when we get the supplies)
  2. Add Taq polymerase.
  3. Aliquot 10uL per PCR tube.
  4. Touch toothpick/pipet tip/loop to colony, then index plate, then swirl around in PCR tube.
  5. 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
  1. 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
REAGENT1 RXN VOLUME (uL)MASTER MIX (uL)
10x rxn buffer2.5x1025
10uM FW primer: G10042x1020
10uM RE primer: G10052x1020
10mM dNTP4x1040
sdH2O14.3x10143
Taq Polymerase0.2x102
DNA---------
Total25x10250
Table 3. PCR Tubes
From Plate (Transformants)To PCR Tubes
2T2Ta
4T4Ta
5T5Ta
6T6Ta
8T8Ta
10T10Ta
11T11Ta
12T12Ta
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 Changes:

  • Aliquot 25uL to PCR tubes
  • Annealing temperature: 63degree C
  • Elongation time: 80s
  • PCR Cycle:
95C @ 2min
Cycle 30x
  1. 95C @ 30s
  2. 63C @ 10s
  3. 72C @ 80s
  4. 72C @ 10min
  5. 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)

Biofilm Track

Eric F. and Jason Gao

Goal

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.

Electroporation Protocol

  1. Prepare overnight culture grown in B2 broth with constant aeration at 37 degrees C
  2. Dilute 1/25 into 25mL of fresh B2 broth in 300mL flask
  3. Grow with constant aeration at 37 degrees C until reading OD 600 of about 0.4

...other steps not shown

Beginning

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