Exeter/31 July 2013

From 2013.igem.org

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(Created page with "== Liquid cultures from the 30/07/2013 == {| class="wikitable" |- ! Liquid culture !! Did it work? |- | K592018 || yes |- | B0015 || yes |- | K592022 || no |- | K864404 || no |-...")
 
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== Liquid cultures from the 30/07/2013 ==
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{{:Team:Exeter/Template/Header}}
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== Liquid cultures from the 30/07/2013 == __NOTOC__
{| class="wikitable"
{| class="wikitable"
Line 37: Line 42:
Half was run on a gel, half was kept aside for ligation.
Half was run on a gel, half was kept aside for ligation.
-
1 - RBS + Cph8 (cut with E + S)
+
1 - RBS + Cph8 (cut with <i>E</i> + <i>S</i>)
-
2 - B0015 (cut with X + P)
+
2 - B0015 (cut with <i>X</i> + <i>P</i>)
3 - negative control (water)
3 - negative control (water)
-
4 -
+
4 - positive control (RFP cut with <i>E</i> + <i>S</i>)
 +
 
 +
5 - positive control (RFP cut with <i>X</i> + <i>P</i>)
 +
 
 +
 
 +
These were then vortexed.
 +
 
 +
 
 +
Master mix
 +
 
 +
120 &micro;l nuclease free water
 +
 
 +
20 &micro;l 10x fast digest buffer w/green.
 +
 
 +
VORTEX.
 +
 
 +
 
 +
15 &micro;l into each PCR tube.
 +
 
 +
Add 5&micro;l DNA
 +
 
 +
Vortex
 +
 
 +
Incubate at 37&deg;C for 10 minutes.
 +
 
 +
== Results of Nanodrop ==
 +
 
 +
 
 +
{| class="wikitable"
 +
|-
 +
! Culture !! Nanodrop concentration (ng/&micro;l) !! Digest (&micro;l)
 +
|-
 +
| 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 <i>E</i> + <i>X</i> (part A)
 +
 
 +
S05058 - cut with <i>E</i> + <i>X</i> (part A)
 +
 
 +
B0015 - cut with <i>E</i> + <i>S</i> (part B)
 +
 
 +
 
 +
== Part A ==
 +
 
 +
250/x = &micro;l DNA needed.            x = Nanodrop value
 +
 
 +
 
 +
250ng DNA
 +
 
 +
2.5&micro;l NEB2 buffer (vortex)
 +
 
 +
0.5&micro;l BSA (vortex)
 +
 
 +
0.5&micro;l <i>EcoRI</i>
 +
 
 +
0.5&micro;l <i>xbaI</i>
 +
 
 +
make up to 20 &micro;l with no nuclease water.
 +
 
 +
 
 +
== Part B ==
 +
 
 +
 
 +
250ng DNA
 +
 
 +
2.5&micro;l NEB2 (vortex)
 +
 
 +
0.5&micro;l BSA (vortex)
 +
 
 +
0.5&micro;l <i>EcoRI</i>
 +
 
 +
0.5&micro;l <i>SpeI</i>
 +
 
 +
make up to 20 &micro;l with no-nuclease water.
 +
 
 +
 
 +
==Vector ==
 +
 
 +
 
 +
250ng plasmid (comes as 25ng/&micro;l)
 +
 
 +
2.5&micro;l NEB2 (vortex)
 +
 
 +
0.5&micro;l BSA (vortex)
 +
 
 +
0.5&micro;l <i>EcoRI</i>
 +
 
 +
0.5&micro;l <i>Pstl</i>
 +
 
 +
0.5&micro;l Dnpl
 +
 
 +
make up to 20 &micro;l with no-nuclease water.
 +
 
 +
 
 +
== Positive controls (<i>E</i> + <i>S</i>) ==
 +
 
 +
 
 +
250ng DNA
 +
 
 +
3.0&micro;l NEB2 (vortex)
 +
 
 +
0.5&micro;l <i>EcoRI</i>
 +
 
 +
0.5&micro;l <i>SpeI</i>
 +
 
 +
make up to 20&micro;l
 +
 
 +
 
 +
== Positive control (<i>X</i> + <i>P</i>) ==
 +
 
 +
 
 +
250ng DNA
 +
 
 +
3.0&micro;l NEB2 (vortex)
 +
 
 +
0.5&micro;l <i>xbaI</i>
 +
 
 +
0.5&micro;l <i>PstI</i>
 +
 
 +
make up to 20&micro;l.
 +
 
 +
 
 +
== Negative control ==
 +
 
 +
 
 +
No DNA
 +
 
 +
0.5&micro;l <i>EcoRI</i>
 +
 
 +
0.5&micro;l <i>SpeI</I>
 +
 
 +
3.0&micro;l NEB2 - vortex
 +
 
 +
make up to 20&micro;l.
 +
 
 +
 
 +
Thermocycler:
 +
 
 +
37&deg;C for 30 minutes
 +
 
 +
80&deg;C for 20 minutes
 +
 
 +
4&deg;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.
 +
 
 +
80&micro;l of RFP, original Nano drop was 12.6ng/&micro;l.
 +
 
 +
 
 +
== Digest gel ==
 +
 
 +
 
 +
Lane 1 - 1kb GeneRuler ladder
 +
 
 +
Lane 2 - RBS + Cph8 (cut with <i>E</i> + <i>S</i>)
 +
 
 +
Lane 3 - B0015 (cut with <i>X</i> + <i>P</i>)
 +
 
 +
Lane 4 - negative control
 +
 
 +
Lane 5 - positive control (RFP, <i>E+S</i>)
 +
 
 +
Lane 6 - positive control (RFP, <i>X+P</i>)
 +
 
 +
Lane 7 - 1kb GeneRuler ladder
 +
 
 +
 
 +
{| class="wikitable"
 +
|-
 +
! 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 ==
 +
 
 +
 
 +
{| class="wikitable"
 +
|-
 +
! 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?
 +
 
 +
Take me back to the [https://2013.igem.org/Team:Exeter/Notebook notebook].
 +
 
 +
  </div>
 +
</div>
 +
{{:Team:Exeter/Template/Footer}}

Latest revision as of 22:12, 1 October 2013

Exeter iGEM 2013 · Paint by Coli

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 - 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

Culture Nanodrop concentration (ng/µl) Digest (µl)
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 = 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


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?

Take me back to the notebook.

Exeter iGEM 2013 · Paint by Coli
Retrieved from "http://2013.igem.org/Exeter/31_July_2013"