Week 36 (2nd-8th of September)

2nd September 2019

1. Yeast Colony PCR

Team members: Selma and Anett

We did colony PCR again with the plates from the 26th July: sample 3,5 and 6.

Procedure

Yeast colony PCR was performed on the following plates that were created on the 22nd of August
3. X3A-.pCCW12-XLHCGR+Ass2A-pPGK1-GPA1-Gαi+Ass2B-pRET2-STE12

5. X3A-GPER+pCCW12-Ass2A-pPGK1-GPA1-Gαi+Ass2B-pRET2-STE1

6. X3A-pccW12-GPER-sfGFP+Ass2A (3-Assembly system)
The colony PCR was conducted as described before (see for example 26th of July, with the difference that the elongation time was 2.5, instead of 1.5 minutes.) with 10 samples (colony11-colony20) of each plate. The PCR product for the 11. colony from all 3 plates were dried after the PCR reaction so we could nut run them on the gel.

Results

Gel: 1kb ladder, negativ control, C12 (plate 3), C13 (plate 3), C14 (plate 3), C15 (plate 3), C16 (plate 3), C17 (plate 3), C18 (plate 3), C19 (plate 3), C20 (plate 3), empty well, C12 (plate 5), C13 (plate 5), C14 (plate 5), C15 (plate 5), C16 (plate 5), C17 (plate 5), C18 (plate 5), C19 (plate 5)

1kb ladder, C20 (plate 5), empty well, C12 (plate 6), C13 (plate 6), C14 (plate 6), C15 (plate 6), C16 (plate 6), C17 (plate 6), C18 (plate 6), C19 (plate 6), C20 (plate 6)

3rd September 2019

On this day we wanted to proceed with the colony PCR, but because some of the samples were missing we couldn’t move on.

1. Setting up E.coli cultures

Team members: Noël and Swenja

Because some Plasmids were running out, we set up ON cultures (37°C, in ca. 5 ml LB+Carb media):

The following constructs were amplified: BAss2, X3C, ZsGreeen, XLHCGR, Ass2C, STE12, Gαi and

4th September 2019

1. Protein plasmid purification of E.coli cultures from 3rd September

Team members: Noël, Swenja and Signe

We did protein purification using the E.Z.N.A Plasmid DNA Mini Kit 1 on the E.coli cultures for yesterday. This was done using the normal procedure.

As we saw no growth in the sample with construct for Gαi we did not do any purification on this sample.