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

120 µl nuclease free water

20 µl 10x fast digest buffer w/green.

VORTEX.

15 µl into each PCR tube.

Add 5µl 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 = µl DNA needed. x = Nanodrop value

250ng DNA

2.5µl NEB2 buffer (vortex)

0.5µl BSA (vortex)

0.5µl EcoRI

0.5µl xbaI

make up to 20 µl with no nuclease water.

Part B

250ng DNA

2.5µl NEB2 (vortex)

0.5µl BSA (vortex)

0.5µl EcoRI

0.5µl SpeI

make up to 20 µl with no-nuclease water.

Vector

250ng plasmid (comes as 25ng/µl)

2.5µl NEB2 (vortex)

0.5µl BSA (vortex)

0.5µl EcoRI

0.5µl Pstl

0.5µl Dnpl

make up to 20 µl with no-nuclease water.

Positive controls (E + S)

250ng DNA

3.0µl NEB2 (vortex)

0.5µl EcoRI

0.5µl SpeI

make up to 20µl

Positive control (X + P)

250ng DNA

3.0µl NEB2 (vortex)

0.5µl xbaI

0.5µl PstI

make up to 20µl.

Negative control

No DNA

0.5µl EcoRI

0.5µl SpeI

3.0µl NEB2 - vortex

make up to 20µl.

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.

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

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.