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/>
== Main purposes today ==
== Main purposes today ==
<hr/>
<hr/>
-
Toxicity of intermediates
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Create dilution series for biolector experiment. 
 +
==Who was in the lab==
 +
<hr/>
 +
Helen, Natalia, Ariadni
-
*25 rxns including duplicates
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==Procedure==
 +
<hr/>
 +
Weighed:
 +
* 2.3187g Ammonium acetate
 +
* 0.2557g Sodium nitrate
 +
* 0.2080g Sodium nitrite
-
*34 rxna including duplicates with the following setup:
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Diluted each in 10mL M9 medium.
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Tube 1-6 containing 5uL pZA21 plasmid template and 3uL of each primers for amplification of the backbone. Tube 7-10; 1uL g-Block from previous purification and 3uL of each Sec signal primers. Tube 11-14 containing same amount of g-Block and primers but now replaced with the TAT2 signal primers. Tube 15-18; 5uL GFP SF plasmid template and 3uL of each primers for GFP SF TAT. Tube 19-22; 5uL GFP SF plasmid template and 3uL of each primers for GFP SF Sec. Tube 23-26; 5uL RFP plasmid template and 3uL of each primer for RFP. Tube 27-30; 1uL g-Block purification and 3uL of each TAT3 primers. Tube 31-34; 0.5uL of the original g-Block and 3uL of each primers for amplifying the g-Block.
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All tube with less than 5uL template DNA was filled with MQ until final volume of 11uL(5+3+3).
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First half of the samples where run on PCR with x7-polymerase and the second half where run with PHUSION-polymerase, with the exception of the g-Block where all reactions where run with PHUSION. This gives following setup:
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Created dilution series as in https://docs.google.com/spreadsheet/ccc?key=0AkM9Z7voM1otdFZ5SWstOWpURGs2S3M5bElMMllkenc
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x7-poly in tube:
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*1-3, 7-8, 11-12, 15-16, 19-20, 23-24, 27-28
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PHUSION in tube:
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*4-6, 9-10, 13-14, 17-18, 21-22, 25-26, 29-30, 31-34
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A mastermix was made for both PCR with standard concentration [https://2013.igem.org/Team:DTU-Denmark/Methods/PCR-mix setup] and one additional volume for pipetting errors:
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Each solution was diluted with media.
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====x7 mastermix for 16rxn====
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<hr/>
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*160uL HF buffer
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*16uL dNTP's 10mM
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*8uL x7 polymerase
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*440uL MQ
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====PHUSION mastermix for 20rxn====
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{| class="wikitable sortable" style="text-align: right"
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! Salt !! mM
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|-
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| Sodium Nitrate || 300
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|-
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| Sodium Nitrate || 30
 +
|-
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| Sodium Nitrate || 3
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|-
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| Sodium Nitrate || 0.3
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|-
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| Sodium Nitrite || 300 (Master)
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|-
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| Sodium Nitrite || 30
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|-
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| Sodium Nitrite || 3
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|-
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| Sodium Nitrite || 0.3
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|-
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| Sodium Nitrite || 0.03
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|-
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| Ammonium Acetate || 3000
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|-
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| Ammonium Acetate || 300
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|-
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| Ammonium Acetate || 30
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|-
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| Ammonium Acetate || 3
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|}
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=208 lab afternoon=
 +
<hr/>
 +
== Main purposes today ==
<hr/>
<hr/>
-
*200uL HF buffer
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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).
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*20uL dNTP's 10mM
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*10uL x7 polymerase
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*5500uL MQ
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-
 
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The tubes were run on two programs simultaneously both where [[Team:DTU-Denmark/Methods/PCR-ramp|ramps]]. First program with the ramp going from 70°C &rarr; 60°C and another from 60°C &rarr; 50°C.
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The tubes in first program(70°C &rarr; 60°C) where tube 7-10, 15-26 and for second program(60°C &rarr; 50°C) tube 1-6, 11-14, 27-34.
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==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}}
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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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