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Exeter/31 July 2013
From 2013.igem.org
Contents |
Liquid cultures from the 30/07/2013
| Liquid culture | Did it work? |
|---|---|
| K592018 | yes |
| B0015 | yes |
| K592022 | no |
| K864404 | no |
| K592011 | no |
| S05058 | yes |
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 - posiive 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
| Culture | Nanodrop concentration (ng/ul) | Digest (ul) |
|---|---|---|
| K592018 | 130.4 | 1.92 |
| B0015 | 81.6 | 3.06 |
| S05058 | 111.0 | 2.25 |
| RFP | 104.4 |
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
| Culture | DNA | NEB2 | Enzyme 1 | Enzyme 2 | Dpnl | No nuclease water | |
|---|---|---|---|---|---|---|---|
| A. RBS + Cph8 | 1.92 | 2.5 | 0.5 | 0.5 | 0.5 | - | 14.08 |
| B. RBS + cyan (S05058) | 2.25 | 2.5 | 0.5 | 0.5 | 0.5 | - | 13.75 |
| C. B0015 | 3.06 | 2.5 | 0.5 | 0.5 | 0.5 | - | 12.94 |
| D. AMP plasmid | 10.0 | 2.5 | 0.5 | 0.5 | 0.5 | 0.5 | 5.5 |
| E. Positive control (RFP, E+S) | 2.39 | 3 | - | 0.5 | 0.5 | - | 13.11 |
| F. Positive control (RFP, X+P) | 2.39 | 3 | - | 0.5 | 0.5 | - | 13.11 |
| G. Negative control (RFP, E+S) | - | 3 | - | 0.5 | 0.5 | - | 16 |
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
| Liquid culture | Did it work? |
|---|---|
| K592018 | yes |
| B0015 | yes |
| S05058 | yes |
| K592022 | no |
| K864404 | no |
| K592011 | no |
The bottom three cultures didnt work again! Could they be on the wrong antibiotic?
