Revision as of 14:46, 2 July 2013

208 lab

Main purposes today

Create dilution series for biolector experiment.

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

Salt	mM
300	30	3	0.3	3000	300	30	3	300 (Master)	30	3	0.3	0.03
2013-07-02	yes	no

Who was in the lab

Henrike, Jakob, Kristian, Gosia

Procedure

PCR for 7 fragments:

Conclusion from today

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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 == Main purposes today ==
 <hr/>
-Toxicity of intermediates
+Create dilution series for biolector experiment.
+Weighed:
+* 2.3187g Ammonium acetate
+* 0.2557g Sodium nitrate
+* 0.2080g Sodium nitrite
-*25 rxns including duplicates
+Diluted each in 10mL M9 medium.
-*34 rxna including duplicates with the following setup:
+Created dilution series as in https://docs.google.com/spreadsheet/ccc?key=0AkM9Z7voM1otdFZ5SWstOWpURGs2S3M5bElMMllkenc
-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.
-All tube with less than 5uL template DNA was filled with MQ until final volume of 11uL(5+3+3).
-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:
+{| class="wikitable sortable" style="text-align: right"
-x7-poly in tube:
+! Salt !! mM
-*1-3, 7-8, 11-12, 15-16, 19-20, 23-24, 27-28
+|-
-PHUSION in tube:
+| Sodium Nitrate | 300
-*4-6, 9-10, 13-14, 17-18, 21-22, 25-26, 29-30, 31-34
+| 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
+|}
-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:
-====x7 mastermix for 16rxn====
-<hr/>
-*160uL HF buffer
-*16uL dNTP's 10mM
-*8uL x7 polymerase
-*440uL MQ
-====PHUSION mastermix for 20rxn====
-<hr/>
-*200uL HF buffer
-*20uL dNTP's 10mM
-*10uL x7 polymerase
-*5500uL MQ
-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.
-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.
 ==Who was in the lab==

Team:DTU-Denmark/Notebook/2 July 2013

From 2013.igem.org