• 13th of June:

Participants: Casper and Kristian K
Purpose: Preparing linearized backbone (pSB1K3) from iGEM biobrick part for USER cloning by means of PCR
Results: Amplification was unsuccessful - no band were detected when running the PCR samples on a 1% agarose gel
A PCR were made to amplify and attach tails for USER cloning to the linearized backbone (pSB1K3) using the primers P1 and P2. Two reactions were prepared according to the table below

• 14th of June:

Participants:
Purpose: Investigating the success of the PCR the day before by gel electrophoresis
Results: PCR amplification were unsuccessful - no bands were detected
PCR products from 130614 was run on a 1% agarose + 1X TAE buffer GEL. 5ul PCR product + 1ul loading dye was loaded on the gel as well as 4ul ladder 1 kb.

• 16th of June:

Participants: Caroline and Anne
Purpose: Optimizing the PCR reaction for amplifying and adding ends for USER cloning to the linearized backbone (pSB1K3 or pSB1C3) by adding DMSO and/or MgCl2 and running a touchdown PCR
Results: PCR amplifications were unsuccessful - no bands were detected when run on gel

PCR optimization were carried out with two different backbones (pSB1K3 and pSB1C3) in different dilutions of template (1x, 5x and 50x) resulting in 6 samples. Each of these samples were prepared according to table 1. Additionally, for each backbone, PCR reactions were made with 2% DMSO, 4% DMSO and 4%DMSO + 1mM MgCl2. The backbone concentration used were diluted twice. The preparation of these samples can be seen in table 2. All PCR samples were run on a touch-down PCR from 53C to 64C A 1% agarose gel were run with PCR samples.

• 18th of June:

Participants: Caroline and Anne
Purpose: Optimizing the PCR reaction for amplifying and adding ends for USER cloning to the linearized backbone (pSB1K3 or pSB1C3) by adding DMSO and/or MgCl2 and running a gradient PCR
Results: Bands of proper lengths were observed when run on a 1% agarose gel for the samples having an annealing temperature of 56.5C and 57.3C

The gradient PCR were carried out on two different samples in 8 replicates using the backbone pSB1C3. The first samples were made with 2% DMSO and the second samples with 4% DMSO and 1mM MgCl2. Preparation of samples were done according to table 1 below. All samples were analysed by means of gel electrophoresis on a 1% agarose gel

• 19th of June:

Participants: Kristian K
Purpose:
Results:

• 24th of June:

Participants: Kristian K, Anne and Caroline
Purpose:
Results:

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A PCR was made to amplify more pSB1C3 backbone with tails for USER cloning to be sure to have enough for the cloning.

• 25th of June:

Participants: Kristian K, Anne and Caroline
Purpose:
Results:

Streaked single colonies of transformants from 140619 as there was no growth on the negative control.

• 30th of June:

Participants: Kristian K, Anne and Caroline
Purpose: Streaking and colony PCR
Results:

A single colony is transferred to 500 μl sterile MilliQ water with an inoculation loop, the same loop is then used to streak on a LB CAM plate.

A PCR mastermix was made as it was the 140613, however, there was not added template to the mastermix, and there was added 0.5 μl Polymerase and 30.5 μl MilliQ water per reaction. 49 μl master mix was transferred to a PCR tube and 1 μl of the dissolved single colony. The PCR was run on the same program as 140613 with annealing temperature of 51 °C.

• 31st of June:

Participants: Kristian K, Anne and Caroline
Purpose:
Results: