Exeter/1 August 2013

From 2013.igem.org

(Difference between revisions)
(Created page with "=== Miniprep === B0015 K592018 S05058 === Nanodrop ===")
Line 9: Line 9:
-
=== Nanodrop ===
+
== Nanodrop ==
 +
 
 +
{| class="wikitable"
 +
|-
 +
! Culture !! Nanodrop concentration (ng/ul) !! for digest (ul)
 +
|-
 +
| B0015 || 30.9 || 8.1
 +
|-
 +
| K592018 || 106.2 || 2.4
 +
|-
 +
| S05058 || 58.2 || 4.3
 +
|-
 +
| RFP || - || -
 +
|}
 +
 
 +
 
 +
== Nanodrop of digests ==
 +
 
 +
We are worried that there is no DNA in our restriction digests, as our gels have no bands.
 +
 
 +
 
 +
=== Group 1 ===
 +
 
 +
Eluted with purite water, digestion by Adam using Victoria's protocol.
 +
 
 +
 
 +
1 - RBS + Cph8 (E+S) - 43.9 ng/ul
 +
 
 +
2 - B0015 (x+p) - 26.7 ng/ul
 +
 
 +
3 - Negative control - 23.3 ng/ul (gives a reading due to NEB2 and BSA)
 +
 
 +
4 - Positive control (E+S) - 37.3
 +
 
 +
5 - Positive control (X+P) - 18.8
 +
 
 +
 
 +
=== Group 2 ===
 +
 
 +
Eluted with RNAse-free water (But possibly contaminated)! Digestion by RAchel and Flick using Victoria's recipe.
 +
 
 +
 
 +
A - RBS + Cph8 (E+X) - 89.3
 +
 
 +
B - RBS + cyan (X+E) - 63.5
 +
 
 +
C - B0015 (E+S) - 66.0
 +
 
 +
D - AMP plasmid (E+P+D) - 40.4
 +
 
 +
E - Positive control (E+S) - 51.5
 +
 
 +
F - Positive control (X+P) - 57.3
 +
 
 +
G - Negative control (E+S) - 33.9 (gives a reading due to NEB2 and BSA)
 +
 
 +
 
 +
== Third retry of digestion ==
 +
 
 +
We have new no-nuclease water from Paul.
 +
 
 +
Instead of using an RFP plasmid from a transformation/mini-prep, we're using one resuspended from a kit plate.
 +
 
 +
 
 +
The reason we're not seeing anything on the gels may be because we're not adding enough DNA.
 +
 
 +
We've split into 2 teams : Clio and Victoria Recipes:
 +
 
 +
 
 +
=== Victoria's Recipe ===
 +
 
 +
{| class="wikitable"
 +
|-
 +
! Culture !! no-nucleotide water !! DNA !! NEB2 !! BSA !! E !! X !! S !! P !!
 +
|-
 +
| 1. RBS + Cph8 || 5.0 || 10 || 2.5 || 0.5 || 1.0 || 1.0 || - || -
 +
|-
 +
| 2. RBS + cyan || 5.0 || 10 || 2,5 || 0.5 || 1.0 || 1.0 || - || -
 +
|-
 +
| 3. B0015 || 5.0 || 10 || 2.5 || 0.5 || 1.0 || - || 1.0 || -
 +
|-
 +
| 5. Positive control (E+S) || 5.0 || 7 || 3 || - || 1.0 || - || 1.0 || -
 +
|-
 +
| 7. Negative control || 15.0 || - || 3 || - || 1.0 || - || 1.0 || -
 +
|}
 +
 
 +
37°C for 10mins, 80°C for 20mins.
 +
 
 +
 
 +
=== Clio's Recipe ===
 +
 
 +
{| class="wikitable"
 +
|-
 +
! Culture !! water !! DNA !! 10x fast buffer w/green !! E !! X !! S !! P !! D !!
 +
|-
 +
| A. RBS + Cph8 || 3 || 10 || 5 || 1.0 || 1.0 || - || - || -
 +
|-
 +
| B. RBS + cyan || 3 || 10 || 5 || 1.0 || 1.0 || - || - || -
 +
|-
 +
| C. B0015 || 3 || 10 || 5 || 1.0 || - || 1.0 || - || -
 +
|-
 +
| D. AMP plasmid || 3 || 7 || 5 || 1.0 || - || - || 1.0 || 1.0
 +
|-
 +
| E. Positive control RFP (X+P) || 5 || 10 || 5 || - || 1.0 || - || 1.0 || -
 +
|-
 +
| G. Negative control || 13 || - || 5 || 1.0 || - || 1.0 || - || -
 +
|}
 +
 
 +
37°C for 30mins , 80°C for 20mins.
 +
 
 +
 
 +
We didnt have much resuspended RFP (hence 7ul DNA used) so 1 each.  We also didt have enough AMP plasmid to do one each.
 +
 
 +
 
 +
===Gel===
 +
 
 +
Lane 1 - 1kb GeneRuler ladder
 +
 
 +
Lane 2 - 1. RBS + Cph8
 +
 
 +
Lane 3 - 2. RBS + Cyan
 +
 
 +
Lane 4 - 3. B0015
 +
 
 +
Lane 5 - 5. RFP control E+S
 +
 
 +
Lane 6 - 7. negative control
 +
 
 +
Lane 7 - A. RBS + Cph8
 +
 
 +
Lane 8 - B. RBS + cyan
 +
 
 +
Lane 9 - C. B0015
 +
 
 +
Lane 10 - D. AMP plasmid
 +
 
 +
Lane 11 - E. RFP control X+P
 +
 
 +
Lane 12 - G. Negative control
 +
 
 +
Lane 13 - 100bp plus DNA ladder.
 +
 
 +
 
 +
Neither worked! No DNA visible.

Revision as of 13:25, 6 August 2013

Contents

Miniprep

B0015

K592018

S05058


Nanodrop

Culture Nanodrop concentration (ng/ul) for digest (ul)
B0015 30.9 8.1
K592018 106.2 2.4
S05058 58.2 4.3
RFP - -


Nanodrop of digests

We are worried that there is no DNA in our restriction digests, as our gels have no bands.


Group 1

Eluted with purite water, digestion by Adam using Victoria's protocol.


1 - RBS + Cph8 (E+S) - 43.9 ng/ul

2 - B0015 (x+p) - 26.7 ng/ul

3 - Negative control - 23.3 ng/ul (gives a reading due to NEB2 and BSA)

4 - Positive control (E+S) - 37.3

5 - Positive control (X+P) - 18.8


Group 2

Eluted with RNAse-free water (But possibly contaminated)! Digestion by RAchel and Flick using Victoria's recipe.


A - RBS + Cph8 (E+X) - 89.3

B - RBS + cyan (X+E) - 63.5

C - B0015 (E+S) - 66.0

D - AMP plasmid (E+P+D) - 40.4

E - Positive control (E+S) - 51.5

F - Positive control (X+P) - 57.3

G - Negative control (E+S) - 33.9 (gives a reading due to NEB2 and BSA)


Third retry of digestion

We have new no-nuclease water from Paul.

Instead of using an RFP plasmid from a transformation/mini-prep, we're using one resuspended from a kit plate.


The reason we're not seeing anything on the gels may be because we're not adding enough DNA.

We've split into 2 teams : Clio and Victoria Recipes:


Victoria's Recipe

Culture no-nucleotide water DNA NEB2 BSA E X S P
1. RBS + Cph8 5.0 10 2.5 0.5 1.0 1.0 - -
2. RBS + cyan 5.0 10 2,5 0.5 1.0 1.0 - -
3. B0015 5.0 10 2.5 0.5 1.0 - 1.0 -
5. Positive control (E+S) 5.0 7 3 - 1.0 - 1.0 -
7. Negative control 15.0 - 3 - 1.0 - 1.0 -

37°C for 10mins, 80°C for 20mins.


Clio's Recipe

Culture water DNA 10x fast buffer w/green E X S P D
A. RBS + Cph8 3 10 5 1.0 1.0 - - -
B. RBS + cyan 3 10 5 1.0 1.0 - - -
C. B0015 3 10 5 1.0 - 1.0 - -
D. AMP plasmid 3 7 5 1.0 - - 1.0 1.0
E. Positive control RFP (X+P) 5 10 5 - 1.0 - 1.0 -
G. Negative control 13 - 5 1.0 - 1.0 - -

37°C for 30mins , 80°C for 20mins.


We didnt have much resuspended RFP (hence 7ul DNA used) so 1 each. We also didt have enough AMP plasmid to do one each.


Gel

Lane 1 - 1kb GeneRuler ladder

Lane 2 - 1. RBS + Cph8

Lane 3 - 2. RBS + Cyan

Lane 4 - 3. B0015

Lane 5 - 5. RFP control E+S

Lane 6 - 7. negative control

Lane 7 - A. RBS + Cph8

Lane 8 - B. RBS + cyan

Lane 9 - C. B0015

Lane 10 - D. AMP plasmid

Lane 11 - E. RFP control X+P

Lane 12 - G. Negative control

Lane 13 - 100bp plus DNA ladder.


Neither worked! No DNA visible.

Retrieved from "http://2013.igem.org/Exeter/1_August_2013"