Week 24 (10th-16th of June)

10th of June

1. DNA extraction

Team members: Benedicte & Claudia To extract DNA from the E. coli liquid cultures of S3C4 & S3C5 from June 6th, Sample 3. pCCW12-GPER-linker-sfGFP from colonies C4 and C5.

Materials

E.Z.N.A. Plasmid DNA Mini Kit I.

Procedure

We took out the tubes from the 37 °C incubator. There was growth in all of them.
We found the miniprep kit in R132: E.Z.N.A. Plasmid DNA Mini Kit I. (For solution I check the fridge in R132)
We vortexed the liquid cultures and pipetted 2 ml into labelled eppendorf tubes. We put the rest (labelled) into the fridge in a plastic bag.
We centrifuged at 10 000 g for 1 min (mistake: we did it at 12 °C instead of room temperature.
We poured out the culture media and kept the pellet.
We added 250 µl Sol I, vortexed the samples, transferred the suspension into new microcentrifuge tubes.
We added 250 µl Sol II, inverted the samples and waited for 2-3 minutes (timing is important!)
We added 350 µl Sol III, inverted the tubes.
We centrifuged the samples at maximum speed for 10 minute. White pellet was formed.
We skipped the optional column equilibration (because of the lack of some solutions).
We transferred the supernatants into columns in 2 ml collection tubes.
We centrifuged it for 1 min at maximum speed, discarded the filtrate.
This time, in contrast to the last time, we were able to find HBC buffer, so we also completed step 8. and 9. of the protocol.
We added 700 µl DNA Wash Buffer, centrifuged it for 30d, discarded the filtrate.
We repeated the previous step again.
We centrifuged the empty column for 2 min at maximum speed to dry the column.
We transferred the column into new microcentrifuge tubes.
We added 50 µl Elution Buffer, let it sit for 1 minute and then we centrifuged it for 1 min at maximum speed.
We did not get any elution (probably because we had to use not the proper column for our experiments). We decided to elute it again (maximum speed but for 5 minutes this time). We got elution!
We ran a gel with 4 µl sample + 1 µl gel red, 5 µl ladder. 100 V for about 35 min. The gel ran a bit long, but samples were not lost. The rest of the sample is in the freezer (labeled: 10-06-2019 Extraction plasmid DNA.

Results

After completing plasmid DNA extraction and purification using the Miniprep kit, we ran a gel and got the following result:

From the left: ladder (1Kb Plus Ladder), S3C4 & S3C5 (pCCW12-GPER-linker-sfGFP)

22th of June

1. Sequencing of sample S3C4 and S3C5

Team members: Claudia and Mads

Aim: The plasmids purified dated 10th June 2019 were sent for sequencing by adding primers to extracted samples.

Materials

Primer
DNA
mH20

Procedure

The concentration of each purified plasmid DNA sample was measured using NanoDrop.
To assure that the whole constructs would be synthesised three primers were used for each vector (USER primer of the first gene and two further primers according to list in R148) Note: We used Sanger sequencing which is limited to the sequencing of approximately 700 bp at a time. Therefore an extra primer was used.
For each construct, three eppendorf tubes had been prepared per sample, one for each of the three primers. The samples were prepared as follows:
- DNA: µl corresponding to 500 ng DNA
- Primer: 3,3 µl
- mH20: x µl
- Total: 10 µl
The stickers with QR codes consists of two parts, the smaller of which was attached into the lab book in a list next to the corresponding construct and primer. The second part was wrapped around the eppendorf tubes in such a way that the QR code was clearly visible.

Data

The list of samples and their concentration and the calculated amount of template to be added for sequencing
Construct	Sample Nr.	ng/µl		µl used
GPER-linker + pCCW12 + sfGFP	S3C5 - 5	60		8,3
GPER-linker + pCCW12 + sfGFP	S3C4 - 4	66,5	63,9	7,7


Construct	Sample Nr.	1. Primer	2. Primer	3. Primer
GPER-linker + pCCW12 + sfGFP	S3C5 - 5	YEA75	YEA137	GPAI-F
GPER-linker + pCCW12 + sfGFP	S3C4 - 4	YEA75	YEA137	GPAI-F

Results

Wrong primers were used for the sequencing so no results.

14th of June

1. Adapted USER ligation of sample 1,2,4 and 6

Team members: Jonas & Swenja

As we haven’t been able to obtain any transformed colonies for sample 4 (XLHGCR-Linker + sfGFP + pCCW12), we had to redo the USER ligation for those. For that, we have received new amplified and purified DNA fragments from Nat, (XLHGCR-Linker and sfGFP). Due to flawed sequences, samples 1 (GPER + pCCW12), 2 (XLHGCR + pCCW12), and 6 (GPAI-Gαs + pPGK1) have also been ligated anew. (Plates from E. coli transformation should not be used again)

Materials

DpnI enzyme
10X USER enzyme
10X CutSmart Buffer
Promoter fragments
Gene fragments
Linearised vector backbones
Per sample:
1. 10x User enzyme 1µl
2. 10x cut smart buffer 1 µl
3. Dpn1 0.5 µl
4. Mili-Q 5.5 µl
5. Vector 1 µl

Procedure

We went to room R-240 (Metabolomics platform laboratory), where you can find a fridge on L.H.S. with a list of REs. There, the enzyme DpnI can be found according to the list. A stock solution was taken and stored in our freezer for later use.
The CutSmart buffer and the USER enzyme have been found in Box no. 6 in the same fridge. We kept a tube of CutSmart buffer for ourselves now and took a stock solution of the USER enzyme which can also be found in our freezer.
Make a total of 10 μL digestion reaction first for each of the samples 1, 2, 4, and 6 as mentioned in the Table below. Each gene with its specific promoter goes into one of the vector backbones and therefore must be digested together.
The final digestion mix is put into the thermocycler PCR machine at;
1. 37 °C for 30 mins
2. 80 °C for 20 mins
3. 25 °C for infinity
Each sample (promoter + gene(s)) is cloned into a different vector, and therefore, once the digested product is ready, 1 μL of linearised vector backbone is added onto each 10 μL reaction digested product samples 1, 2, 4, and 6 based on their respective backbone as mentioned in the table below. The final mix of linearised vector backbone added to the digested product sample was then put in the thermocycler PCR machine for step-down cooling for the ligation reaction to occur at;
1. 37 °C for 10 mins
2. 25 °C for 10 mins
3. 20 °C for 10 mins
4. 12 °C infinity
We use a 5-assembler system for genomic integration of the genes.
1. X3A: Promoter pCCW12 with receptor
2. Ass 2A: Promoter pPGK1 with GPA1-Gαs
The vector backbones are linearised before mixing with the promoter and gene fragment mix. The vector backbones of the USER cassette are digested with restriction enzymes AsiSI and NbBsmI that cut at specific sites in the backbone that linearises the backbone and creates sticky ends that are specific for our digested sequences (promoter + gene fragment) to bind.