Team:UCL/Labbook/Week17

Bacterial Labs

Monday 23rd September

Nanodrop readings

Tuesday 24th September

CMV PCR with Taq polymerase

-10 reactions and 1 control

Tubes were labelled from 1 to 11. This was unsuccessful.

Made new inoculations from 5X CMP and 1X CMP plates for Ligated CMV-MMP9.

Sunday 29th September

There were not enough colonies for B3&4. The rest of the samples were minipreped and digested and run on a gel cut.

4 more inoculations in 10 ml LB broth of A2, A5, B2 and C2. Nanodrop readings of these inoculations were poor.

Mammalian Labs

Monday 23rd September

Seeded 2x105 cells (1x105 cells/ml) per well into 16 wells set in three 6-well plates. Stock Hela passaged (P21).

24th September

CMV PCR with Taq polymerase

-10 reactions and 1 control

This was unsuccessful.

25th September

Aim: To stably transfect Hela cells with ’’Str Ble’’.

Confluency of dishes: 30-50%

Per well of the 6-well plates: 13 well total.

1. Mass of DNA: 2.0µg

2. Volume of DNA dissolved in TE Buffer: 5.0 µl

3. Final volume of DNA diluted in serum free media: 100 µl

4. Volume of superfect (SF) reagent:10.0 µl

5. Volume of serum-containing media: 600 µl

2x ”mock transfected” wells (No DNA, No SE)

Procedure:

Made a ”mask mix” solution for the 13 well-plates to be transfected, with the following composition :

1. 34 ng

2. 93.5 ul

3. 1870 ul

4. 187 ul

5. 11220 ul

Transfer DNA-Buffer solution to 15 ml falcon tube. Add non-serum media (1776.5µl), Filter, Add SF, Vortex (10.6), Incubate for 5-10 min at room temperature. Meanwhile aspirate and wash cells with 4ml PBS. Add media with serum (600ml). Pipette up and down x2. Apply to cells(≈700µl). Incubate for 2-3 hours. Change media. Incubate (24 hours)

Note : for characterisation , add 1µl of 100µg/ml zeocin to one plate, 2µl to another, and 3µl to the last to make up the concentrations to those over the page.

26th September

Characterisation:

24 hours after exposure:

Sunday 29th September