Team:Exeter/lab book/3gip/wk8

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     <!------------ENTER LAB BOOK HERE: PLEASE INCLUDE DATES WITHIN THE WEEK------------>  
+
     <p><b><u>Tuesday 28th August</u></b></p>
 +
<p><i><b>Morning</b></i></p>
 +
<p>•<u>3A assembly</u></p> 
 +
<p>BBa_B0034 + WbnJ in pSB1K3</p>
 +
<p>BBa_B0034 + WbnK in pSB1K3</p>
 +
<p>BBa_B0034 + WfcA in pSB1K3</p>
 +
<p>BBa_B0034 + WbbC(1) in pSB1K3</p>
 +
<p>BBa_B0034 + WbbC(2) in pSB1K3</p>
 +
<p>BBa_B0034 + WbbC(3) in pSB1K3</p>
 +
<p>BBa_B0034 + WclY in pSB1K3</p>
 +
<p>BBa_B0034 + WbiP in pSB1C3</p>
 +
<p>BBa_B0034 + WbiP in pSB1C3</p>
 +
<p>Performed with the NEB protocol with some adaptions again. The volume of digestion was changed from 50ul to 20ul by adding less water so as to increase the DNA concentration. The enzymes and buffer concentrations were adapted as below. </p>
 +
<p>
 +
</p>
 +
<p><i><b>Afternoon</b></i></p>
 +
<p>•<u>Transformation:</u> </p>
 +
<p>Chloramphenicol plasmid backbone pSB1C3, 2012 Distribution Kit Plate 1 3A</p>
 +
<p>Tetracycline plasmid backbone pSB1T3, 2012 Distribution Kit Plate 1 7A</p>
 +
<p>Kanamycin plasmid backbone pSB1K3, 2012 Distribution Kit Plate 1 5A</p>
 +
<p>Ampicillin plasmid backbone pSB1A3, 2012 Distribution Kit Plate 1 1G</p>
 +
<p>Using Invitrogen TOP10 competent cells split into 2 eppendorfs containing 25μl each. </p>
 +
<p>Spread on the relevant antibiotic plates for each transformation using 150μls. </p>
 +
 
 +
<p><b><u>Wednesday 29th August</u></b></p>
 +
<p><i><b>Morning</b></i></p>
 +
 
 +
<p>•<u>Gel Digest of DNA from 21/08/2012</u></p>
 +
<p>Lanes 1, 2, 3, 4 ompA</p>
 +
<p>Lanes 6, 7, 8, 9  BBa_J23119 + BBa_B0034_WbnJ</p>
 +
<p>Lanes 10, 11 BBa_J13002 + WbnK_BBa_B0014v
 +
<p>
 +
</p>
 +
<p>Planned another 3A assembly but had run out of plasmid backbone. </p>
 +
<p><i><b>Afternoon</b></i></p>
 +
<p>•<u>Adding cultures</u></p> 
 +
<p>Cells transformed with pSB1C3, pSB1T3, pSB1K3, pSB1A3 were added into liquid medium and incubated overnight. </p>
 +
<p>Protocol was followed using the relevant antibiotic for the BioBrick part as the selection antibiotic. </p>
 +
<p><b><u>Thursday 30th August</u></b></p>
 +
<p><i><b>Morning</b></i></p>
 +
<p>•<u>Mini-Prepping</u> of pSB1C3, pSB1T3, pSB1K3, pSB1A3</p>
 +
 
 +
<p>pSB1C3, pSB1T3, pSB1K3 and pSB1A3 were nanodropped and recorded very good concentrations. </p>
 +
 
 +
<p><i><b>Afternoon</b></i></p>
 +
<p>•<u>Transformation</u></p>
 +
<p>The ligations were transformed using the Competent Cell protocol and using the Competent Cells made in the lab. </p>
 +
<p>•<u>Adding cultures</u></p> 
 +
<p>Cells transformed with Cyclodextrin in pSB1C3, hyaluronan synthase in pSB1C3, cyclodextrin + BBa_B0014 in pSB1C3, hyaluronan synthase + BBa_Boo14 in pSB1C3, sacB + BBa_Boo14 in pSB1C3 were added into liquid medium and incubated overnight. See, Showcasing Polysaccharide Production: 27th - 31st August 2012, Thursday 30/08/12</p>
 +
<p>Protocol was followed using chloramphenicol for the BioBrick part as the selection antibiotic. </p>
 +
 
 +
<p><b><u>Friday 31st August</u></b></p>
 +
<p><i><b>Morning</b></i></p>
 +
<p>•<u>3A assembly</u> </p>
 +
<p>BBa_J13002 + WbnJ_BBa_B0014 in pSB1K3</p>
 +
<p>BBa_J13002 + WbnK_BBa_B0014 in pSB1K3</p>
 +
<p>BBa_K764001+ WfcA_BBa_B0014 in pSB1A3</p>
 +
<p>BBa_K764001+ WclY_BBa_B0014 in pSB1A3</p>
 +
<p>BBa_K026000 + BBa_B0034 in pSB1K3</p>
 +
<p>BBa_K026001 + BBa_B0034 in pSB1K3</p>
 +
<p>BBa_J23119 + BBa_B0034 in pSB1K3</p>
 +
<p>BBa_I0050 + BBa_B0034 in pSB1K3</p>
 +
<p>BBa_B0034_WbbC obtained from PCR into pSB1C3</p>
 +
<p>BBa_B0034 in pSB1A3</p>
 +
<p>BBa_J13002 in pSB1A3</p>
 +
<p>Performed with the NEB protocol with some adaptions again. The volume of digestion was changed from 50μl to 20μl by adding less water so as to increase the DNA concentration. The enzymes and buffer concentrations were adapted as below. This was not a Linearized Plasmid Backbone so for the plasmid digestion we used the NEB Biolabs protocol adapted to the amounts shown below. </p>
 +
<p>
 +
</p>
 +
<p><i><b>Afternoon</b></i></p>
 +
<p>•<u>Transformation</u></p>
 +
<p>The ligations were transformed using the Competent Cell protocol and using the Competent Cells made in the lab. </p>
 +
      
      
     </font>
     </font>

Revision as of 16:04, 25 September 2012

ExiGEM2012 Lab Book 3GP wk6

The 3-Gene Inducible Plasmid: 27th - 31st August 2012

Tuesday 28th August

Morning

3A assembly

BBa_B0034 + WbnJ in pSB1K3

BBa_B0034 + WbnK in pSB1K3

BBa_B0034 + WfcA in pSB1K3

BBa_B0034 + WbbC(1) in pSB1K3

BBa_B0034 + WbbC(2) in pSB1K3

BBa_B0034 + WbbC(3) in pSB1K3

BBa_B0034 + WclY in pSB1K3

BBa_B0034 + WbiP in pSB1C3

BBa_B0034 + WbiP in pSB1C3

Performed with the NEB protocol with some adaptions again. The volume of digestion was changed from 50ul to 20ul by adding less water so as to increase the DNA concentration. The enzymes and buffer concentrations were adapted as below.

Afternoon

Transformation:

Chloramphenicol plasmid backbone pSB1C3, 2012 Distribution Kit Plate 1 3A

Tetracycline plasmid backbone pSB1T3, 2012 Distribution Kit Plate 1 7A

Kanamycin plasmid backbone pSB1K3, 2012 Distribution Kit Plate 1 5A

Ampicillin plasmid backbone pSB1A3, 2012 Distribution Kit Plate 1 1G

Using Invitrogen TOP10 competent cells split into 2 eppendorfs containing 25μl each.

Spread on the relevant antibiotic plates for each transformation using 150μls.

Wednesday 29th August

Morning

Gel Digest of DNA from 21/08/2012

Lanes 1, 2, 3, 4 ompA

Lanes 6, 7, 8, 9 BBa_J23119 + BBa_B0034_WbnJ

Lanes 10, 11 BBa_J13002 + WbnK_BBa_B0014v

Planned another 3A assembly but had run out of plasmid backbone.

Afternoon

Adding cultures

Cells transformed with pSB1C3, pSB1T3, pSB1K3, pSB1A3 were added into liquid medium and incubated overnight.

Protocol was followed using the relevant antibiotic for the BioBrick part as the selection antibiotic.

Thursday 30th August

Morning

Mini-Prepping of pSB1C3, pSB1T3, pSB1K3, pSB1A3

pSB1C3, pSB1T3, pSB1K3 and pSB1A3 were nanodropped and recorded very good concentrations.

Afternoon

Transformation

The ligations were transformed using the Competent Cell protocol and using the Competent Cells made in the lab.

Adding cultures

Cells transformed with Cyclodextrin in pSB1C3, hyaluronan synthase in pSB1C3, cyclodextrin + BBa_B0014 in pSB1C3, hyaluronan synthase + BBa_Boo14 in pSB1C3, sacB + BBa_Boo14 in pSB1C3 were added into liquid medium and incubated overnight. See, Showcasing Polysaccharide Production: 27th - 31st August 2012, Thursday 30/08/12

Protocol was followed using chloramphenicol for the BioBrick part as the selection antibiotic.

Friday 31st August

Morning

3A assembly

BBa_J13002 + WbnJ_BBa_B0014 in pSB1K3

BBa_J13002 + WbnK_BBa_B0014 in pSB1K3

BBa_K764001+ WfcA_BBa_B0014 in pSB1A3

BBa_K764001+ WclY_BBa_B0014 in pSB1A3

BBa_K026000 + BBa_B0034 in pSB1K3

BBa_K026001 + BBa_B0034 in pSB1K3

BBa_J23119 + BBa_B0034 in pSB1K3

BBa_I0050 + BBa_B0034 in pSB1K3

BBa_B0034_WbbC obtained from PCR into pSB1C3

BBa_B0034 in pSB1A3

BBa_J13002 in pSB1A3

Performed with the NEB protocol with some adaptions again. The volume of digestion was changed from 50μl to 20μl by adding less water so as to increase the DNA concentration. The enzymes and buffer concentrations were adapted as below. This was not a Linearized Plasmid Backbone so for the plasmid digestion we used the NEB Biolabs protocol adapted to the amounts shown below.

Afternoon

Transformation

The ligations were transformed using the Competent Cell protocol and using the Competent Cells made in the lab.