Exeter/31 July 2013

Liquid cultures from the 30/07/2013

We then mini-prepped the liquid cultures that worked.

Mini Prep

We centrifuged the entire falcon tube for 10 minutes at 5000 rpm.

Then resuspended the whole pellet.

Nuclease free water was then run through instead of elution buffer, this was done twice.

Digest

Half was run on a gel, half was kept aside for ligation.

1 - RBS + Cph8 (cut with E + S)

2 - B0015 (cut with X + P)

3 - negative control (water)

4 - positive control (RFP cut with E + S)

5 - positive control (RFP cut with X + P)

These were then vortexed.

Master mix

120ul nuclease free water

20ul 10x fast digest buffer w/green.

VORTEX.

15ul into each PCR tube.

Add 5ul DNA

Vortex

Incubate at 37°C for 10 minutes.

Results of Nanodrop

Restriction Digest - Victoria's Recipe

We are attaching terminators (B0015) to K592018 (RBS+Cph8) and S05058 (RBS+cyan). We are putting them in the AMP plasmid.

K592018 - cut with E + X (part A)

S05058 - cut with E + X (part A)

B0015 - cut with E + S (part B)

Part A

250/x = ul DNA needed. x = Nanodrop value

250ng DNA

2.5ul NEB2 buffer (vortex)

0.5ul BSA (vortex)

0.5ul EcoR1

0.5ul xba1

make up to 20ul with no nuclease water.

Part B

250ng DNA

2.5ul NEB2 (vortex)

0.5ul BSA (vortex)

0.5ul EcoR1

0.5ul Spel

make up to 20ul with no-nuclease water.

Vector

250ng plasmid (comes as 25ng/ul)

2.5ul NEB2 (vortex)

0.5ul BSA (vortex)

0.5ul EcoR1

0.5ul Pstl

0.5ul Dnpl

make up to 20ul with no-nuclease water.

Positive controls (E + S)

250ng DNA

3.0ul NEB2 (vortex)

0.5ul EcoR1

0.5ul Spel

make up to 20ul

Positive control (X + P)

250ng DNA

3.0ul NEB2 (vortex)

0.5ul xba1

0.5ul Pstl

make up to 20ul.

Negative control

No DNA

0.5ul EcoR1

0.5ul Spe1

3.0ul NEB2 - vortex

make up to 20ul.

Thermocycler:

37°C for 30 minutes

80°C for 20 minutes

4°C hold

Add water and DNA first, then enzymes, buffer and BSA.

Make sure NEB and BSA are completely defrosted and vortex before using.

Vortex and spin down before using the thermocycler.

- Had to do a sure clean on the RFP.

80ul of RFP, original Nano drop was 12.6ng/ul.

Digest gel

Lane 1 - 1kb GeneRuler ladder

Lane 2 - RBS + Cph8 (cut with E + S)

Lane 3 - B0015 (cut with X + P)

Lane 4 - negative control

Lane 5 - positive control (RFP, E+S)

Lane 6 - positive control (RFP, X+P)

Lane 7 - 1kb GeneRuler ladder

Ran the gel:

Lane 1 - 1kb Geneladder

Lane 2 - A

Lane 3 - B

Lane 4 - C

Lane 5 - D

Lane 6 - E

Lane 7 - F

Lane 8 - G

Lane 9 - ladder

The gel failed , again. Ladders ran but otherwise there was no DNA present.

Remade liquid cultures

The bottom three cultures didnt work again! Could they be on the wrong antibiotic?

Take me back to the notebook.