Team:DTU-Denmark/Notebook/2 July 2013

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{{:Team:DTU-Denmark/Templates/StartPage|02 July 2013}}
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Navigate to the [[Team:DTU-Denmark/Notebook/1_July_2013|Previous]] or the [[Team:DTU-Denmark/Notebook/3_July_2013|Next]] Entry
=208 lab=
=208 lab=
<hr/>
<hr/>
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Create dilution series for biolector experiment.   
Create dilution series for biolector experiment.   
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==Who was in the lab==
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<hr/>
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Helen, Natalia, Ariadni
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==Procedure==
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<hr/>
Weighed:
Weighed:
* 2.3187g Ammonium acetate
* 2.3187g Ammonium acetate
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Created dilution series as in https://docs.google.com/spreadsheet/ccc?key=0AkM9Z7voM1otdFZ5SWstOWpURGs2S3M5bElMMllkenc
Created dilution series as in https://docs.google.com/spreadsheet/ccc?key=0AkM9Z7voM1otdFZ5SWstOWpURGs2S3M5bElMMllkenc
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Each solution was diluted with media. 
{| class="wikitable sortable" style="text-align: right"
{| class="wikitable sortable" style="text-align: right"
! Salt !! mM
! Salt !! mM
|-
|-
-
| Sodium Nitrate | 300
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| Sodium Nitrate || 300
-
| Sodium Nitrate | 30
+
-
| Sodium Nitrate | 3
+
-
| Sodium Nitrate | 0.3
+
-
| Ammonium Acetate | 3000
+
-
| Ammonium Acetate | 300
+
-
| Ammonium Acetate | 30
+
-
| Ammonium Acetate | 3
+
-
| Sodium Nitrite | 300 (Master)
+
-
| Sodium Nitrite | 30
+
-
| Sodium Nitrite | 3
+
-
| Sodium Nitrite | 0.3
+
-
| Sodium Nitrite | 0.03
+
|-
|-
-
| [[Team:DTU-Denmark/Notebook/2_July_2013|2013-07-02]] || yes || no
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| Sodium Nitrate || 30
 +
|-
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| Sodium Nitrate || 3
 +
|-
 +
| Sodium Nitrate || 0.3
 +
|-
 +
| Sodium Nitrite || 300 (Master)
 +
|-
 +
| Sodium Nitrite || 30
 +
|-
 +
| Sodium Nitrite || 3
 +
|-
 +
| Sodium Nitrite || 0.3
 +
|-
 +
| Sodium Nitrite || 0.03
 +
|-
 +
| Ammonium Acetate || 3000
 +
|-
 +
| Ammonium Acetate || 300
 +
|-
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| Ammonium Acetate || 30
 +
|-
 +
| Ammonium Acetate || 3
|}
|}
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=208 lab afternoon=
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<hr/>
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== Main purposes today ==
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<hr/>
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Performing [[Team:DTU-Denmark/Methods/Colony_PCR|Colony PCR]] to verify if transformants contain proper construct (signal peptide, GFP, RFP - in plazmid pZA21).
==Who was in the lab==
==Who was in the lab==
<hr/>
<hr/>
-
Henrike, Jakob, Kristian, Gosia
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Gosia, Ariadni
==Procedure==
==Procedure==
<hr/>
<hr/>
-
[[Team:DTU-Denmark/Methods/PCR|PCR]] for 7 fragments:
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Among colonies which grew after transformation 11 colonies were chosen to be verified by colony PCR.
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Plates with transformants and colonies checked by colony PCR are signed:  
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== Conclusion from today ==
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# T2 U2 Sec  - colonies 01, 02, 03
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<hr/>
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# T2 U2 TAT2 - colonies 01, 02, 03
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Inconsistent results from the first PCR where obtained with bands in some of the reactions but not in the duplicates. Even the g-Block was not seen though we have easily amplified that before with very bright bands. Something must have gone wrong and that's why we made the additional 34rxns which will be analyzed on gels tomorrow.
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# T2 U3 TAT3 - colonies 01, 02, 03, 1a, 2a
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T2 U2 Sec stands for 2nd transformation, 2nd USER reaction, Sec signal peptide
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TAT2 and TAT3 is TAT signal peptide
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<hr/>
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All checked colonies were plated on a new plate, overnight incubation at 37°C.
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Navigate to the [https://2013.igem.org/Team:DTU-Denmark/Notebook/25_June_2013 Previous] or the [https://2013.igem.org/Team:DTU-Denmark/Notebook/27_June_2013 Next] Entry
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PCR master mix for 12 samples (one additional just in case) was done according to [[Team:DTU-Denmark/Methods/Colony_PCR|Colony PCR]] method. Forward primer for colonies with Sec signal (PCR tubes signed: 1, 2, 3) peptide was primer for GFP Sec FW (5a). Forward primer for colonies (PCR tubes signed: 4, 5, 6, 7, 8, 9, 10, 11) with TAT was GFP TAT FW (4a). Reversed primer for all colonies was RFP RW (6b n).
 +
 
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In positive colonies amplified fragment of lenght about 1550 bp is expected.
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PCR program (IGEM_CO)
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# 98°C for 2:00 
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# 98°C for 0:10
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# 67°C for 0:05
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#* ramp 0.1°C/s until 62°C
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# 72°C for 1:30
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# Go to number 2 - repeat 50
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# 72°C for 5:00
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# 4°C for 10:00:00
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# End
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 +
 
 +
 
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Navigate to the [[Team:DTU-Denmark/Notebook/1_July_2013|Previous]] or the [[Team:DTU-Denmark/Notebook/3_July_2013|Next]] Entry
{{:Team:DTU-Denmark/Templates/EndPage}}
{{:Team:DTU-Denmark/Templates/EndPage}}

Latest revision as of 11:14, 4 October 2013

02 July 2013

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Contents

208 lab

Main purposes today

Create dilution series for biolector experiment.

Who was in the lab

Helen, Natalia, Ariadni

Procedure

Weighed:

  • 2.3187g Ammonium acetate
  • 0.2557g Sodium nitrate
  • 0.2080g Sodium nitrite

Diluted each in 10mL M9 medium.

Created dilution series as in https://docs.google.com/spreadsheet/ccc?key=0AkM9Z7voM1otdFZ5SWstOWpURGs2S3M5bElMMllkenc

Each solution was diluted with media.

Salt mM
Sodium Nitrate 300
Sodium Nitrate 30
Sodium Nitrate 3
Sodium Nitrate 0.3
Sodium Nitrite 300 (Master)
Sodium Nitrite 30
Sodium Nitrite 3
Sodium Nitrite 0.3
Sodium Nitrite 0.03
Ammonium Acetate 3000
Ammonium Acetate 300
Ammonium Acetate 30
Ammonium Acetate 3

208 lab afternoon

Main purposes today

Performing Colony PCR to verify if transformants contain proper construct (signal peptide, GFP, RFP - in plazmid pZA21).

Who was in the lab

Gosia, Ariadni

Procedure

Among colonies which grew after transformation 11 colonies were chosen to be verified by colony PCR. Plates with transformants and colonies checked by colony PCR are signed:

  1. T2 U2 Sec - colonies 01, 02, 03
  2. T2 U2 TAT2 - colonies 01, 02, 03
  3. T2 U3 TAT3 - colonies 01, 02, 03, 1a, 2a

T2 U2 Sec stands for 2nd transformation, 2nd USER reaction, Sec signal peptide TAT2 and TAT3 is TAT signal peptide

All checked colonies were plated on a new plate, overnight incubation at 37°C.

PCR master mix for 12 samples (one additional just in case) was done according to Colony PCR method. Forward primer for colonies with Sec signal (PCR tubes signed: 1, 2, 3) peptide was primer for GFP Sec FW (5a). Forward primer for colonies (PCR tubes signed: 4, 5, 6, 7, 8, 9, 10, 11) with TAT was GFP TAT FW (4a). Reversed primer for all colonies was RFP RW (6b n).

In positive colonies amplified fragment of lenght about 1550 bp is expected.

PCR program (IGEM_CO)

  1. 98°C for 2:00
  2. 98°C for 0:10
  3. 67°C for 0:05
    • ramp 0.1°C/s until 62°C
  4. 72°C for 1:30
  5. Go to number 2 - repeat 50
  6. 72°C for 5:00
  7. 4°C for 10:00:00
  8. End


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