Team:Freiburg/Notebook/1 August

From 2011.igem.org

Revision as of 14:46, 9 September 2011 by UsernameV (Talk | contribs)


This is the wiki page
of the Freiburger student
team competing for iGEM 2011.
Thank you for your interest!

Contents

green light receptor

Qickchange PCR of CcaS

Investigators:Julia
repeating experiment from___, because we had no positive transformation, probably chosen wrong annealing temperature before.

blue light receptor

PCR

Investigators: Sophie


As our last PCRs of the LovTAP with the Gibson overhangs didn't work well, we now try a PCR with primers without overhangs for Gibson assembly.


Name: Sophie


Date: 01.08.11
Continue from Experiment: PCR (Date): 27.07.11

(Name): Sandra, Sophie

Project Name: Blue Light

PCR-Mixture for one Reaction:

For a 50 µl reaction use


32,5µl H20 Name
10µl 5x Phusion Buffer of Primer
2.5µl Primer fw P35
2.5µl Primer dw P36
1µl dNTPs of Template DNA
1µl DNA-Template M35 (LovTAP)
0.5 µl Phusion (add in the end)

What program do you use?

LovTAP ohne Ueberhaenge


To confirm the PCR-Product has the correct size, load 2 µl of the sample onto an agarose-gel.

caption

How did you label the PCR-Product, where is it stored and what do you do next?

Labelled: M35 short; stored in Gibson Stuff box.

red light receptor

3A-assembly with pcyA and the terminator BBa_1006

Investigators:Julia

1.Digestion:

terminator (S21a), 26µl of the miniprep+ 12,1 µl H2O, cut with XbaI&PstI
pcyA (part BBa_I15009), 3 µl of miniprep + 35µl H2O, cut with EcoRI&SpeI
Vector-backbone (10 µl) pSB1T3, cut with EcoRI,PstI & DpnI

to each reation 5µl BSA(10x) and 5µl NEB buffer4 were added, digested at 37°C for two hours, inactivated at 80°C for 20min.

2.Ligation


2µl of digested pcyA,terminator and vector were added to 11µl H2O,2µl ligase buffer and 1µl T4 ligase.
Incubated at room temperature for 35 min, inactivated at 80°C for 20 min.

3.Transformation

Lysis cassette

Phage Lysis Cassette (K124017) + RBS (B0034)

Investigators: Theo


Digestion of 3A Assembly


Name: Theo Date: 01.08.2011
Continue from Experiment : -
Project Name: Phage Lysis Cassette (K124017) + RBS (B0034)

Procedure


  1. add H2O (38μl-DNA )
  2. 5 μl NEB4 buffer (stored at iGEM’s, -20°C)
  3. 5 μl 10x BSA (used 1:10 diluted sample stored at iGEM’s, -20°C)
  4. DNA (500 ng) (Vector:Insert ratio 1:3 in following Ligation)
  5. 1 μl restriction enzymes (stored at iGEM’s, -20°C)
  6. heat for 1-2 hours 37°C (6 hours if time)
  7. heat for 20 minutes 80°C (inactivation of enzymes)
  8. keep at 4°C if you cannot continue

Restriction enzymes you need to cut the vector, insert1 and insert 2:


Components
Vector (μl)
Insert1 -B0034- and 2 -K124017-(μl)
DNA (500ng) 10 9 8
BSA (10x) (5μl)
NEB4 Buffer (5μl)
Enzyme 1 (1μl) EcoRI + DpnI EcoRI XbaI
Enzyme 2 (1μl) PstI SpeI PstI
H2O (38 μl- DNA) 27 31 30
In total 50 μl

Documentation:

Why are you doing this experiment? Where are the samples stored? Antibiotica resistance, vector used etc.


A RBS (ribosome binding site) between the lamba cI regulated Promotor (R0051 of K098995) and the holin ATG of K124017 is absent. So a 3A assembly had to be performed in order to get a functioning Lysis cassette.

Resistance of RBS (B0034)=Cm and of Phage Lysis Cassette (K124017)=Amp, so the Vector is Tet (pSB1T3)



Ligation


Name: Theo Date: 01.08.2011
Continue from: 01.08.2011 Phage Lysis Cassette (K124017) + RBS (B0034) Digestion
Project Name: Phage Lysis Cassette (K124017) + RBS (B0034) Ligation

Procedure


PCR tube:

total volume 20 μl


  1. add H2O (17 μl -X-Y-Z)
  2. add 2 μl Ligase Buffer 10x
  3. add Insert 1, Insert 2(when proceeding from 3A digestion use 2 μl of each)
  4. add Vector (20ng needed. When proceeding from 3A digestion use 2 μl)
  5. Add 1 μl T4-DNA Ligase
  6. Incubate 10-30 min at room temperature
  7. heat for 20 minutes at 80°C
  8. store at -20°C or directly proceed to transformation


Name of part Ratio Insert:Vector

= 3:1 or 1:1

Volume (μl)
X insert 1 Modif. K098995 3:1 (1*3) 3
Y insert 2 K124017 3:1 (2*3) 6
Z vector pSB1T3 1:3 (1) 1
H2O 7

Documentation:

Why are you doing this experiment? Where are your parts stored? Name the parts for ligation etc.


Ligation step of 3A assembly.


How to calculate ratios --> e.g. for K124017

Length of pSB1T3= ca 2200bp

Length of K124017+Vector= ca 4500bp

4500/2200= ca 2

So 2*3 µl (since 3:1 is needed) = 6 µl





Transformation


Name: Theo Date:
Continue from : 26.07.2011 Lysis Cassette V.2 Ligation



Project Name: Lysis Cassette V.2 (3A assembly of quickchange-modified K098995 with Lysis genes K124017)

Procedure


  1. take cells from -80°C freezer and put them on ice! (every eppi contains about 400 μl cells)
  2. thaw cells on ice 20 minutes
  3. pipette 50 μl cells and 2 μl DNA into eppi still on ice!
  4. Incubate for 30 minutes on ice
  5. Heat at 42°C for 60 sec
  6. Incubate on ice for 5 minutes
  7. Add 200 μl LB Broth
  8. Incubate for 2 hours at 37°C (cells with lysis cassette at 30°C!!)
  9. Plate 50 μl and 200μl on two different LB/Agar plates with appropriate antibiotic resistance

Documentation:

Why are you doing this experiment? Name of the sample? Where are they stored? Name the vector with inserts, antibiotika resistance etc.


New Composite Part in pSB1T3 Vector with quickchange-modified K098995 and Lysis genes K124017



Precipitator

PCR


Name: Sophie


Date: 01.08.11
Continue from Experiment: replay of PCR 19.07.11 (Date) 19.07.11

(Name) Ruediger

Project Name: GFP Pbd

PCR-Mixture for one Reaction:

For a 50 µl reaction use


32,5µl H20 Name
10µl 5x Phusion Buffer of Primer
2.5µl Primer fw P28
2.5µl Primer dw P18 / P19/ P20
1µl dNTPs of Template DNA
1µl DNA-Template S 14 (GFP)
0.5 µl Phusion (add in the end)

What program do you use?

Ruediger PCR (modified by Tobi and me. First 10 cycles with 55°C, next cycles with 62°C annealing temperature.

To confirm the PCR-Product has the correct size, load 2 µl of the sample onto an agarose-gel. capture

How did you label the PCR-Product, where is it stored and what do you do next?

Retrieved from "http://2011.igem.org/Team:Freiburg/Notebook/1_August"