5/7/2016

Preparation of agar plates with Kanamycin + Chloroamphenol.

A) LB agar: require 37g/L. We measured 14.8g for 400mL water. Then autoclaved. (Needs to be cool enough to touch, not too hot though 45-55°C)

B) Addition of antibiotics: only add once cool enough to touch. Working concentration is a 1000x dilution of stock concentrations. i.e. from mg/mL to μg/mL.

C) Pour plates - around 25mL/plate. (top tip: use pipette gun to suck up bubbles). Performed preparation of chemically competent cells (specifically TOP10 and BL21). Using protocol specified with the following alteraions: spin for 20 mins rather than 10. Bacterial transformation (chemical) (with Lubas DNA- we are checking that the cells are inded competent as well as their transformation efficiency). Using bacterial transformation (chemical) protocol with the following alterations. Heat shock for 1 minute. Incubated at 37°C for 1 hour.

Transformed one of each cell type with different plasmid DNA:

TOP10 + KT244 TAm 1n pET29a
TOP10 + pQR801 w-TAm
BL21 + KT244 TAm 1n pET29a
BL21 + pQR801 w-TAm

06/07/16

The transformations worked

07/07/16

Abbie + Kuba + Dale plated out 4 glycerol stock biobricks:

3 on Chloramphenicol:
BBa_M30109 pSB1AC3 6/12/13
BBa_K812014 pAB1C3 7/26/13
BBa_i712004 pSB1AC3 7/26/13

BBa_E0015 pSB1AK3 8/7/121

8/07/16

Abbie + Kuba + Dale Checked colonies and made Ampicillin plates. However the colonies did not grow over night.

1. Made Ampicillin, Chloramphenicol and Kanamycin stock solutions as indicated in Table 1. above.
500/125/250mg of each respectively to 5mL of water or ethanol in the case of chloramphenicol.
2. Poured plates: approx 25mL.
2* chloramphenicol (50mL LB + 50μg Chlor. stock)
15* Amp.(350mL LB + 350μg Amp. stock)
3. Plate spreading:
Kanamycin positive control.
BBa_1712004 pSB1AC3
BBa_J63008 / BBa_J63008 AmpK
BBa_B0015 pSB1AK3
BBa_K5120 pSB1C3
4. Falcon tube inoculation: addition of 5μL of antibiotic
BBa_1715004 pSB1AC3 (Chloramphenicol + control)

BBa_J63008/BBa_J63009 AmpR (Ampicillin + control)

BBa_B0015 pSB1AK3 (Kanamycin + control

BBa_K5126 pSB1c3 (Chloramphenicol + control)

11/07/16

10-12:30PM : Abbie + Kuba + Dale analysed samples incubated over the weekend.

12/07/16

Perform the miniprep

13/07/16

Today we have inoculated 4x samples to get pSB1C3, pSB1K3, pSB1A2, pSB1A3 backbones. Dale and Amandeep have minipreped 2x cultures (from the day before) in the afternoon. If today’s inoculations are successful, we hope to do more minipreps tomorrow morning

20/07/16

Dale had prepared some agar and media

08/09/2016

Today was an exciting day as we prepared our DNA to be shipped and become part of the registry. We prepared our samples according to protocol and dried them overnight. We then performed mini preps of RBS + GFP in order from the distribution kits (2016 and 2013) in order to perform a restriction digest with our pH sensitive promoter GadA. and created glycerol stocks so we can begin experimenting on these constructs soon.

Team:UCL/amandeep/Experiments

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