Transformations, Digestions and Reverse Transcriptions.

12/01/2010 -

Using the ligations Tracey created over the Christmas holidays further transformations were set up using the DHS-Alpha bacteria, following the same protocol as previously. The agar plates were then incubated overnight at 37 degrees Celsius.

13/01/2010 -

Lots of colonies grew on the 1 µl and 3 µl plates but not as many on the 5 µl plate. It was decided to pick colonies from the 1 µl and 3 µl plates but not the 5 µl plates as these colonies did not work as well when Tracey did the transformation, but they will be left as a back up. 5 colonies were picked from each plate. These were then incubated in LB + 5 µl ampicillin overnight at 37 degrees Celsius, shaking overnight.

14/01/2010 -

DNA purification using a quick method which does not use columns.
This method is used when there are a lot of samples (10 in my case) as the QIAgen mini-prep method is expensice, by not using columns it makes it cheaper. The protocol is as follows:
1) Take 1.5ml culture and spin at 13,000rpm for 2 minutes to form a pellet.
2) Remove supernatant
3) Add 100 µl of P1 resuspension buffer
4) Resuspend pellet very gently.
5) Add 200 µl of P2 buffer
6) Add 150 µl of P3 buffer
7) Invert 6 times
8) Centrifuge at 13,000rpm for 3 minutes
9) Add supernatant to 1ml of 100% ethanol (in clean eppendorf tubes)
10) Vortex
11) Centrifuge at 13,000 rpm for 10 minutes
12) Remove ethanol, use a smaller pipette to remove dregs
13) Leave to air dry
14) Resuspend pellet in 40 µl water

Then two digests were set up using H3 and Sac1 restriction enzymes.

1 µl DNA
1 µl enzyme buffer
1 µl enzyme
7 µl water

These digestions were incubated at 37 degrees Celsius for 1 hour. Once the digestion was completed the samples were run on an agarose electrophoresis gel. The image revealed none of the vectors had taken up the insert.

15/01/2010 - 17/01/2010 -

Over the weekend Tracey set up a number of mini-preps using the colonies I had grown previously. These were purified using the no columns method then digested using Sac1. After running on an electrophoresis gel it was revealed no insert had been taken up.

Tracey then re-ligated the vectors which had contained the insert in the wrong orientation. The DNA was purified and digested with Sac1. Again it was seen that insert has been incorporated, but in the wrong orientation. This may have been because not all the initial vector had been re-ligated and this was what was coming through on the gel.

18/01/2010 -

Reverse Transcription - creation of cDNA for use in a Taqman MicroRNA assay.

Tracey had previously extracted RNA from SW1353 and HeLa cells, both of which are Cancer cell lines (SW1353= Human Bone Chondrosarcoma, HeLa= Cervical Carcinoma)

This RNA was very concentrated so was diluted 1 in 5 (5 µl RNA in 25 µl water). The nanodrop was then used to check the diluted concetration:

SW1353 = 711.8 ng/ µl
HeLa = 6.18 ng/ µl

This was still very concentrated so the RNA was diluted again

SW1353 = 1.4 µl in 7.6 µl water
HeLa = 1.6 µl in 7.4 µl water

This gave a final volume of 9 µl

For each cell line 2 tubes were set up one which will use random hexamers and one which would use miR-34 primers.

To the diluted RNA 2 µl of either random hexamers or miR-34 primers was added. The tubes were then incubated at 70 degrees Celsius for 10 minutes, using a hot block.

While the tubes were incubating a 5x mastermix of reverse transcription agents was made up. By making this mastermix it helps with precision of pipetting and helps to keep the volume of each agent pipetted in each tube constant, helping with accuracy.

On ice add : -

1x 5x
4 µl 5 x 1st strand buffer 20 µl
2 µl DTT 10 µl
1 µl dNTPs 5 µl
1 µl Superscript RT 5 µl
1 µl RNase Inhibitor 5 µl

9 µl of the mastermix was added to each tube
These tubes were then incubated at 42 degrees Celsius for 1 hour then at 70 degrees Celsius for 10 minutes
The tubes were then stored at -20 degrees Celsius.

Tracey picked 12 colonies and incubated then in LB + 5 µl ampicillin overnight at 37 degrees Celsius, shaking, overnight.