Team:Freiburg/Notebook/19 July

From 2011.igem.org

(Difference between revisions)
(→Theoretical Gibson-Assembly)
(→Precipitator)
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===NAME OF YOUR EXPERIMENT===
===NAME OF YOUR EXPERIMENT===
 +
'''PCR'''
-
'''Investigators: NAME'''
+
 
 +
{| style="border-spacing:0;"
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Name: Ruediger
 +
 
 +
 
 +
 
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Date: 19.07.11
 +
 
 +
|-
 +
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Continue from Experiment (Date)
 +
 
 +
PCR 18.07 (Name)
 +
 
 +
|-
 +
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Project Name: GFP Pbd
 +
 
 +
|}
 +
PCR-Mixture for one Reaction:
 +
 
 +
For a 50 µl reaction use
 +
 
 +
 
 +
{| style="border-spacing:0;"
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 32,5µl
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| H<sub>2</sub>0
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Name
 +
 
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 10µl
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 5x Phusion Buffer
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| of Primer
 +
 
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 2.5µl
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Primer fw
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| P18, P19, P20
 +
 
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 2.5µl
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Primer dw
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| P28
 +
 
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 1µl
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| dNTPs
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| of Template DNA
 +
 
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 1µl
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| DNA-Template
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| PCR product of P1,P3,S14 from yesterday
 +
 
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 0.5 µl
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Phusion (add in the end)
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
 
 +
|}
 +
What program do you use?
 +
 
 +
One set of probes was prepared and then split into 3 tubes each to test them at Annealingtemperatures 44C, 52C and 60C for the first 10 cycles. Then Annealingtemperature 60C for 25 cycles
 +
 
 +
 
 +
To confirm the PCR-Product has the correct size, load 2 µl of the sample onto an agarose-gel.
 +
 
 +
 
 +
How did you label the PCR-Product, where is it stored and what do you do next?
 +
 
 +
S14+P18+P28
 +
 
 +
S14+P19+P28
 +
 
 +
S14+P20+P28
 +
 
 +
Stored in PCR product box
 +
 
 +
[[File:Freiburg11_7_20_2011_2_48_01_PM.Jpg]]
 +
Lane1
 +
Quick Load Marker
 +
Lane2
 +
44C S14+P18+P28
 +
Lane3
 +
44C S14+P19+P28
 +
Lane4
 +
44C S14+P20+P28
 +
Lane5
 +
52C S14+P18+P28
 +
Lane6
 +
52C S14+P19+P28
 +
Lane7
 +
52C S14+P20+P28
 +
Lane8
 +
60 S14+P18+P28
 +
Lane9
 +
60 S14+P19+P28
 +
Lane10
 +
60 S14+P20+P28

Revision as of 16:32, 19 July 2011

Contents

green light receptor

NAME OF YOUR EXPERIMENT

Investigators:NAME


blue light receptor

Theoretical Gibson-Assembly

Investigators: Sandra, Sophie

Primer Degin for Blue light sensor (lovTAP) + trp promotor (BBa_K322999) and tetR Gen + tetO promotor (BBa_Q04400).

  • LOVtap-Trp_up: gaattcgcggccgcttctagtcacacaggaaagtactatgt
  • LOVtap-Trp_dw: tacttttatctaatctggacatctagtatttctcctctttgtcgataccctttttacgtg
  • TetR-TetO_up: aaagaggagaaatactagatgtccagattag
  • TetR-TetO_dw^: ctgcagcggccgctactag

^we also have another primer for this one, but we are not sure about the length/melting temperature. Might be changed.

red light receptor

NAME OF YOUR EXPERIMENT

Investigators:NAME



Lysis cassette

NAME OF YOUR EXPERIMENT

Investigators:NAME


Precipitator

NAME OF YOUR EXPERIMENT

PCR


Name: Ruediger


Date: 19.07.11
Continue from Experiment (Date)

PCR 18.07 (Name)

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 P18, P19, P20
2.5µl Primer dw P28
1µl dNTPs of Template DNA
1µl DNA-Template PCR product of P1,P3,S14 from yesterday
0.5 µl Phusion (add in the end)

What program do you use?

One set of probes was prepared and then split into 3 tubes each to test them at Annealingtemperatures 44C, 52C and 60C for the first 10 cycles. Then Annealingtemperature 60C for 25 cycles


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


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

S14+P18+P28

S14+P19+P28

S14+P20+P28

Stored in PCR product box

Freiburg11 7 20 2011 2 48 01 PM.Jpg Lane1 Quick Load Marker Lane2 44C S14+P18+P28 Lane3 44C S14+P19+P28 Lane4 44C S14+P20+P28 Lane5 52C S14+P18+P28 Lane6 52C S14+P19+P28 Lane7 52C S14+P20+P28 Lane8 60 S14+P18+P28 Lane9 60 S14+P19+P28 Lane10 60 S14+P20+P28

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