Ligations, Plating, Western Blots and Taqman

23/11/09 -

Agar plates were made up by melting agar which then had 100 microL ampicillin added. The agar + amp was poured into petri-dishes and left to set.

While the agar plates were setting a transformation for each ligation was set up as follows: -
- Add 10 microL ligation to 50 microL DHSalpha (competent bacteria which had been treated with calcium chloride)
- Leave on ice for 15 minutes
- Place in 40 degrees Celsius water bath for 1 minute
- Place on ice for 2 minutes
- Add 500 microL LB
- Incubate at 37 degrees Celsius for 45 minutes, shaking.
- Spin for 2 minutes at 13,000 rpm
- Remove most of the LB, leave about 100 microL
- Resuspend very gently
- Pipette onto agar plate and spread with sterile spreader
- Incubate over night at 37 degrees Celsius.

24/11/09 -

No colonies had grown on the vector only agar plates and only a few grew on the other plates.

These few colonies were picked and added with LB with 100 microL ampicillin added.
These were incubated over night at 37 degrees Celsius, shaking.

25/11/09 -

Another digest was carried out as before. The digested vectors were then phenol chloroform cleaned as before. The vector was then run on an electrophoresis gel to check it was present.

The band was cut out of the gel and purified using the same method as previously.

26/11/09 -

Worked in cell culture today with Alba Warn to passage my cells at a particular density. It was decided plating 300,000 cells per well was sufficient. By plating at a known density it will allow me to determine how many population doublings have occurred between each passage.

The cells were counted using a Hemocytometer.

Results:

65 cells over 5 sections of the hemocytometer
65/5 = 13
13 x 10,000 = 130,000
So, 130,000 cells per ml
Cells were resuspend in 5ml so, 650,000 cells in total.

Need to plate 300,000 cells so need to take 2.3ml and transfer to new flask. This allowed for two new flasks to be passaged into P.31. The final volume was made up to 10ml using medium.

The remaining P.30 cells were passaged to P.31 and will be left to become confluent. When the cells are confluent they will be harvested and frozen down in case the cells become infected again.

The other flask of P.30 cells was used to harvest protein and RNA as follows:

- Aspirate medium
- Wash with PBS
- Trypsinise
- Incubate for a few minutes
- Resuspend cells in 1ml PBS
- Transfer 500 microL into two eppendorfs (one for RNA extraction, one for protein extraction)
- Spin at 13,000 rpm for 5 minutes

The protein extraction was carried out as before. For the RNA extraction the pellet was resuspended in 500 microL of TRIzol and frozen for the extraction to be completed another day.

Another ligation was set up as follows: -

Ligation	Insert	Vector	Ligase	Buffer	Water
	μL
1	2	1	2	2	13
2	5	1	2	2	10
3	10	1	2	2	5
4	0	1	2	2	15

The complimentary sequence for miR-34a also arrived this week so we are now able to set up a positive control for the ligations

Firstly they need to be made into DNA, which will be treated the same way as a PCR product. This was done as follows:

- Add 10 microL of forward oligo to 10 microL of reverse oligo and 30 microL water
- Boil on hot block for 5 minutes
- Leave to cool naturally (this allows for strands to anneal and create DNA)