Week 6: 22^nd July - 28^th July

Monday, July 22nd

We did a PCR on the seven products of golden gate 1 using the VR and VF2 primers. We mixed :

• 10 uL of One Taq Buffer 10X
• 1 uL of 10 mM dNTPs
• 1 uL of each VF2 and VR primers
• 35,5 uL of H2O
• 1 uL of the One Taq enzyme
• 1 uL of the golden gate products

We used the following PCR program :

1. 95°C for 3 min
2. 95°C for 15 sec
3. 55°C for 30 sec
4. 68°C for 2 min
5. 68°C for 10 min

Note: We repeated step 2, 3 and 4 29 times.

The gel migration didn't reveal any band. We supposed that it hadn't worked due to the annealing temperature we used. Indeed, with the help of the New England BioLabs Tm Calculator website, we have found that the optimal annealing temperature for those primers was 53°C.

Tuesday, July 23rd

In order to test the best annealing temperature for our following PCRs, we did a PCR on our 5 first golden gate products with two different annealing temperature : 53 or 51°C. We also prepared two positiv controls for each conditions with either bacteria transformed by empty PSB1A3 plasmid or 1 uL of the miniprep plasmid. This was realised in order to verify if the problem of our first PCR was due to a bad lysis of our bacteria. What is more, we prepared one negativ control with an annealing temperature of 55°C.

(Mettre photo gel)

According to our results, we can assume that our first PCR was badly realised due to a handling error and not because of the annealing temperature we used.

In the meantime, we did the Golden Gate 1 again in order to obtain better ligation results. We transformed TOP 10 E. coli with the golden gate products and plated them on LB-Agar with carbenicillin antibiotic. The petri dishes have been let overnight at 37°C.

Wednesday, July 24th

We did a colony PCR from colony obtained on the petri dishes plated with golden gate 2 products transformed bacteria. We chose one white colony of each of the first ten different FUR BS constructions. The positiv control was prepared by using the PSB1A3 plasmid. The same PCR program was used but we were out of One Taq so we utilize Dream Taq.

We re isolate colony of bacteria transformed with our golden gate 1 products.

Thursday, July 25th

We did a 1% gel in order to migrate our PCR products. It revealed that our PCR hadn't work. We then realised that we hadn't changed the elongation temperature of our PCR program. Indeed, the dream taq functions at 72°C.

We did a PCR colony with the white colony obtained after we plated our golden gate 1 products transformed bacteria.

We used the following PCR program :

1. 95°C for 3 min
2. 95°C for 15 sec
3. 55°C for 30 sec
4. 72°C for 2 min
5. 72°C for 10 min

(mettre photo gel)

Considering our gel, we can reasonably think that our golden gate had worked properly. We then did miniprep.

Friday, July 26th

We used nanodrop to measure the concentration of plasmid of our minipreps. The concentrations obtained were extremely low. We decided to do it again, but we got the same low results.

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