Team:Freiburg/Notebook/1 August

From 2011.igem.org

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were digested with XbaI and PstI.
were digested with XbaI and PstI.
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====2.Ligation====
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'''Investigators: Theo'''
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Ligation of:
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*S39+P18
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*S43+P18
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*S39+P19
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*S43+P19
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*S39+P20
 +
*S43+P20
 +
 +
stored in Theo´s box.

Revision as of 12:52, 13 September 2011


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 29 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 B0034 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: 01.08.2011
Continue from : 01.08.2011 Phage Lysis Cassette (K124017) + RBS (B0034) Ligation



Project Name: Phage Lysis Cassette (K124017) + RBS (B0034)

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 modified K124017 containing a strong RBS in front of first ATG

RESULT: No colonies!!! :( Probably because of the Tet vector, since other members of the group also had problems. We are trying it again with the pSB1C3 Vector



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?


3A-assembly

1.Digestion

Investigators: Theo

Digestion of PCR product.

  • S39
  • S43

were digested with EcoRI and SpeI

  • P18
  • P19
  • P20

were digested with XbaI and PstI.


2.Ligation

Investigators: Theo

Ligation of:

  • S39+P18
  • S43+P18
  • S39+P19
  • S43+P19
  • S39+P20
  • S43+P20

stored in Theo´s box.

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