10/6/2014

Safety Instructions

The team received mandatory safety training and general lab routine information.

A really cool video about Chalmers' chemical safety routine (recorded at our very own lab!) can be seen in http://www.chalmers.se/insidan/EN/about-chalmers/environmental-work/policies/chemical-safety-routine

17/6/2014

Introduction to centrifuges and autoclaves

As a part of the mandatory training required by the SysBio group, the team received instructions on how to operate the centrifuges and autoclaves of the labs.
We also worked on our risk declaration; a mandatory document containing our experiments descriptions and information about the chemicals we are going to use, their handling, storage and associated risk, as well as waste handling.

20/6/2014

Finished mandatory training

Our Risk Declaration was approved by the lab manager and our supervisors. We also sign a safety certificate stating that we are aware of the safety instructions and with that, completed the mandatory "check-in list for new incomers" of the lab.

2/6/2014

Media Preparation

On the first day of laboration, we prepared LB, YPD and SC-Ura/His/Trp-Leu and YPD media.
Click here to see the protocol. [[PUT METHOD]]!
[[File:20140623_103124.jpg|frameless|300px]],[[File:20140623_115809.jpg|frameless|300px]]

2/6/2014

Amplification and Purification of non-synthetic parts

Succesfully PCR amplified five out of the seven parts from yeast genome via a Phusion High-Fidelity DNA Polymerase. Click here to see the protocol.
The PCR program used was:
1. 3 min at 98ºC
2. 10s at 98ºC
3. Gradient from 52 to 61ºC for 30s
4. 1 min at 72ºC
5. Go to step 2 (repeat 39 times)
6. 10 min at 72ºC.
A diagnostic gel was performed and the result was as follows: [[File: Bio-Rad 2014-06-25 15hr 34min.jpg|frameless|300px]]
The purification of the succesfully amplified parts was done using a PCR Purification Kit from GenJet following the instructions of the manufacture's manual but with no addition of isopropanol.

10/6/2014

Plasmid Purification

Performed plasmid purification on E. coli cells containing plasmids p413, p414, p415, p416, p2055 and a plasmid containig our Cas9 sequence (pTPG1-dCas9-UPGP)via a GenJet Plasmid Purification Kit. The manufacter's instructions were followed, except the elution buffer was subtituted by water on the last step.

Amplification and purification of parts

Performed a PCR amplification of the non-synthetic parts that failed on the day before and on the dCas9 sequence and the three fluorecent proteins -Yellow, Cyano and Red (YFP, CFP and RFP, respectively).
The PCR was done using the Phusion High-Fidelity DNA Polymerase (see protocol here) and the program used was:
1. 3 min at 98ºC
2. 10s at 98ºC
3. Gradient from 50 to 55ºC for 30s
4. 30s at 60ºC
5. 2min 15s at 72ºC
5. Go to step 2 (repeat 39 times)
6. 10 min at 72ºC.
A diagnostic gel was performed and the result is as follows:
[[File: Bio-Rad 2014-06-26 17hr 53min.jpg|frameless|300px]]