Team:Cambridge/LabBook/Week10

From 2010.igem.org

(Difference between revisions)
(New page: <div align="center">Week 10: Monday 13th September - Sunday 19th September</div> ==Monday== ===100. Expt: Adding pBAD to P.P. pSB1C3 (Hannah and Emily)=== *Took P.P. pSB1C3 out of fridge ...)
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|20µl
|20µl
|}
|}
 +
 +
2. Mix gently and spin down
 +
 +
3. Incubate at 37°C in a heat block or water thermostat for 5min (we actually left them for 30 mins). Optional: Inactivate the enzyme by heating for 5min at 80°C.
 +
 +
====Fermentas Ligation Protocol====
 +
1. Add the following to a microcentrifuge tube:
 +
{|class="wikitable"
 +
|-
 +
|Linearized vector DNA
 +
|10-100ng
 +
|-
 +
|Insert DNA (at 3:1 Molar excess over vector)
 +
|variable
 +
|-
 +
|5x Rapid Ligation Buffer
 +
|4µl
 +
|-
 +
|T4 DNA Ligase, 5u/µl
 +
|1µl
 +
|-
 +
|Water, nuclease-free
 +
|to 20µl
 +
|}
 +
 +
2. Vortex and spin briefly to collect drops.
 +
 +
3. Incubate the mixture at 22°C (RT) for 5 mins (we used 30 mins)
 +
 +
4. Use 2-5µl of the ligation mixture for transformation
 +
 +
The reaction mixture can be stored at 0-4°C until used for transformation.
 +
 +
===101. Expt: Transformation of plasmid obtained in experiment on p88===
 +
 +
We transformed TOP10 cells with the plasmid extracted from the experiment on p88 using the standard protocol. We incubated overnight on Cm plates and obtained colonies on plates from plasmids of lanes: bottom; 2 top; 8; 5; 7; 8; 9.
 +
 +
We then grew overnight LB cultures for plasmid extraction.
 +
 +
The colonies had a slightly different aspect suggesting some possible contamination. I've therefore extracted plasmids from 3 lanes:
 +
*Lane 1 bottom: 19.8ng/µl
 +
*Lane 2 top: 5.6ng/µl
 +
*Lane 5: 16.8ng/µl
 +
 +
These 3 have been sent to sequencing.
 +
 +
===102. Expt: Making glycerol stock===
 +
In CC iGEM 2010 Green Box in -80°C Freezer:
 +
 +
1. DNA2.0 PP in TOP10 from source
 +
 +
2. DNA2.0 LC in TOP10 from source
 +
 +
3. PP + pSB1C3
 +
 +
4. LC + pSB1C3
 +
 +
5. LC + pBAD + pSB1C3
 +
 +
6. Thio Lane 1 bottom
 +
 +
7. Thio Lane 2 top
 +
 +
8. Thio Lane 5
 +
 +
9. Thio Lane 7
 +
 +
==Tuesday==
 +
===103. Expt: Mutagenesis of Luciola cruciata===
 +
Will's mutagenesis primers will all be sorted in the green box in the freezer once diluted. Ordered primers diluted to 100µM as suggested by Biolegio.
 +
 +
Working stocks will be made (to reduce freeze-thaw damage to the main stocks) at 10mM.
 +
 +
Primers in reactions should be at 0.5µM (approx) so 1µl of dilute primer stock per 20µl reaction.
 +
 +
====Colony PCR====
 +
*Lyse cells first, take a loopful of colony and add 20µl of ddH20 (in a PCR tube)
 +
*Heat at 98°C for 10 mins
 +
*Cool to -80°C for ~10 mins
 +
*Vortex for ~2mins
 +
 +
PCR mix:
 +
{|class="wikitable"
 +
|-
 +
|ddH20
 +
|7µl
 +
|-
 +
|Phusion Mast Mix (2x)
 +
|10µl
 +
|-
 +
|F Primer
 +
|1µl
 +
|-
 +
|R Primer
 +
|1µl
 +
|-
 +
|Lysate
 +
|1µl
 +
|-
 +
|
 +
|20µl reactions
 +
|}
 +
 +
Used mutagenesis.f.LC and mut286.r.LC --> Labelled (1)
 +
 +
and mutagenesis.r.LC and mut286.f.LC (to mutate back to wild type) --> Labelled (2)
 +
 +
Started Gibson assembly with (1) and (2), products from colony PCR with 15µl Gibson master mix, 2.5µl (1), 2.5µl (2)
 +
 +
===104. Expt: Adding pBAD to P.P. pSB1C3 (Hannah + Emily)===
 +
µl°C
µl°C

Revision as of 11:54, 1 October 2010

Week 10: Monday 13th September - Sunday 19th September

Contents

Monday

100. Expt: Adding pBAD to P.P. pSB1C3 (Hannah and Emily)

  • Took P.P. pSB1C3 out of fridge
  • Extracted pBAD
  • Restricted pBAD with E + S (using protocol from Fermentas below)
  • Restricted P.P. pSB1C3
  • Ran on gel

Here are gel columns:

Lane 1 Lane 2 Lane 3 Lane 4 Lane 5 Lane 6 Lane 7
EZ Ladder II P.P.pSB1C3 P.P.pSB1C3 pBAD (1) pBAD (1) pBAD (2) pBAD (2)
5µl 24µl 12µl 12µl 12µl 12µl 12µl

Results of gel: P.P.+pSB1C3 worked but pBAD could not be seen. P.P.+pSB1C3 was around 5kb as expected. Ben performed gel extraction on this.

We used pBAD and P.P.+pSB1C3 from a previous experiment (from the freezer) and performed ligation using the Fermentas protocol below, but using the following quantities:

Volume (µl)
P.P. pSB1C3 1
pBAD 1
DI H20 13
5x Rapid ligation Buffer 4
T4 DNA Ligase 1
20

Fermentas Restriction Digest Protocol

1. Combine the following reaction components at room temperature in the order indicated:

Plasmid DNA
Water, nuclease-free 15µl
10x FastDigest Buffer 2µl
DNA 2µl (up to 1µg)
FastDigest enzyme 1µl
20µl

2. Mix gently and spin down

3. Incubate at 37°C in a heat block or water thermostat for 5min (we actually left them for 30 mins). Optional: Inactivate the enzyme by heating for 5min at 80°C.

Fermentas Ligation Protocol

1. Add the following to a microcentrifuge tube:

Linearized vector DNA 10-100ng
Insert DNA (at 3:1 Molar excess over vector) variable
5x Rapid Ligation Buffer 4µl
T4 DNA Ligase, 5u/µl 1µl
Water, nuclease-free to 20µl

2. Vortex and spin briefly to collect drops.

3. Incubate the mixture at 22°C (RT) for 5 mins (we used 30 mins)

4. Use 2-5µl of the ligation mixture for transformation

The reaction mixture can be stored at 0-4°C until used for transformation.

101. Expt: Transformation of plasmid obtained in experiment on p88

We transformed TOP10 cells with the plasmid extracted from the experiment on p88 using the standard protocol. We incubated overnight on Cm plates and obtained colonies on plates from plasmids of lanes: bottom; 2 top; 8; 5; 7; 8; 9.

We then grew overnight LB cultures for plasmid extraction.

The colonies had a slightly different aspect suggesting some possible contamination. I've therefore extracted plasmids from 3 lanes:

  • Lane 1 bottom: 19.8ng/µl
  • Lane 2 top: 5.6ng/µl
  • Lane 5: 16.8ng/µl

These 3 have been sent to sequencing.

102. Expt: Making glycerol stock

In CC iGEM 2010 Green Box in -80°C Freezer:

1. DNA2.0 PP in TOP10 from source

2. DNA2.0 LC in TOP10 from source

3. PP + pSB1C3

4. LC + pSB1C3

5. LC + pBAD + pSB1C3

6. Thio Lane 1 bottom

7. Thio Lane 2 top

8. Thio Lane 5

9. Thio Lane 7

Tuesday

103. Expt: Mutagenesis of Luciola cruciata

Will's mutagenesis primers will all be sorted in the green box in the freezer once diluted. Ordered primers diluted to 100µM as suggested by Biolegio.

Working stocks will be made (to reduce freeze-thaw damage to the main stocks) at 10mM.

Primers in reactions should be at 0.5µM (approx) so 1µl of dilute primer stock per 20µl reaction.

Colony PCR

  • Lyse cells first, take a loopful of colony and add 20µl of ddH20 (in a PCR tube)
  • Heat at 98°C for 10 mins
  • Cool to -80°C for ~10 mins
  • Vortex for ~2mins

PCR mix:

ddH20 7µl
Phusion Mast Mix (2x) 10µl
F Primer 1µl
R Primer 1µl
Lysate 1µl
20µl reactions

Used mutagenesis.f.LC and mut286.r.LC --> Labelled (1)

and mutagenesis.r.LC and mut286.f.LC (to mutate back to wild type) --> Labelled (2)

Started Gibson assembly with (1) and (2), products from colony PCR with 15µl Gibson master mix, 2.5µl (1), 2.5µl (2)

104. Expt: Adding pBAD to P.P. pSB1C3 (Hannah + Emily)

µl°C

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