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From 2010.igem.org

Week-7

Preparation for getting the bricks:

(1)Competent cell preparation

(2)Transformation of the Biobricks into DH5-alpha competent cells

(3)Verificiation of the lengths of the biobricks by Gel Electrophoresis after PCR.

Lab Setup

We prepared the Competent cells to transform the Bio-bricks obtained from the registry.

Preparation of competent cells

1.Start with preparing competent cells protocol

2.Pour out the 250mL cultivation solution

250/6 = 41.88 ≈ 42mL

3.Separate into 6 falcon tubes ×42mL

4.3000 g centrifugation at 4℃for 10 mins

5.Liquid taken away from supernatant

6.Then, cells in each tube was resuspended with 1mL CCMB80 buffer

Media was prepared and plated with appropriate Antibiotics.

LB medium

800mL H2O

16g LB-broth powder

 Adjust the medium to pH= 7.49

LB agar medium

800mL H2O

 12g agar

16g LB-broth powder

 Adjust the medium to pH= 7.51

SOC-mechice

20g trypton

 5h yeast extract

 0.5g NaCl

 0.372g KCl

   7.2g Glucose

 400mL H2O

 Separated half of the mixture in to 2 bottles and added 475mL of H2O in each bottle, which makes the total volume of 950mL. Ampicillin stock preparation

Tube weights 13.28747g

Tube with ampicillin weights 16.65591g

Amipicillin weights 3.36844g

Target concentration 100mg/mL=0.1g/mL

Dissolved in 33.684mL H2O and mixed well. Filtered with 0.22μm filter and poured 1mL in each tube. Frozen the tubes in -8 degree freezer.

Agar plate preparation with ampicillin

Amipicillin stock concentration=100mg/mL

Volume of agar medium=800mL

Ampicillin concentration required = 0.1mg/mL

Mamp=800mL × 0.1mg /mL= 80mg

Vsoc, with amp= 80mg /100mg/mL= 0.8mL in the 800mL SOC medium

The colonies obtained were amplified by colony PCR. (Refer to the PCR protocol for further details). The PCR product was run on a GEL to verify whether the Bio-bricks are of correct lengths. Having verified the lengths of the Bio-bricks the selected colonies were inoculated over night. The inoculated colonies were extracted.

Details of the biobricks we are using: