Team:Nevada/Week 18

From 2012.igem.org

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[[Team:Nevada/Week 19| Week 19]]  
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[[Team:Nevada/Week 19| Week 19]] |
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[[Team:Nevada/Final Weeks| Final Weeks]] |
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:::purify protein from 10, 10ml tubes
:::purify protein from 10, 10ml tubes
:::incubate with rice for 5 hours
:::incubate with rice for 5 hours
 +
==September 18==
==September 18==
 +
Joe:    RFP-SBP ligation for submission
Joe:    RFP-SBP ligation for submission
     culture more RFP-SBP in Top10 cells
     culture more RFP-SBP in Top10 cells
Line 42: Line 45:
Michelle:
Michelle:
 +
:::Small cultured two successful colonies from M2-BL21 transformation with LB AMP-CM.
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    Small cultured two successful colonies from M2-BL21 transformation with LB AMP-CM.
 
 +
:Justin and Dafne:
 +
:::Express more SBP-B12 protein
 +
:::Using 96-well binding plate, add 100 ul of concentrated protein to 10 wells
 +
:::incubate overnight
-
Justin and Dafne
 
-
Express more SBP-B12 protein
 
-
    Using 96-well binding plate, add 100 ul of concentrated protein to 10 wells
 
-
    incubate overnight
 
==September 19==
==September 19==
-
Joe:    Transform RFP-SBP for submission
+
:Joe:    Transform RFP-SBP for submission
Justin and Dafne
Justin and Dafne
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Michelle:
Michelle:
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     PCR colony check M2-BL21 with Forward Primer (SBP-sense124)(10x diluted) and Reverse Primer (antilysine)(10x diluted) before large scale culturing brightest colony with 100ml LB AMP-CM and making glycerol stocks.
+
     PCR colony check M2-BL21  
 +
    Forward Primer (SBP-sense124)(10x diluted) and Reverse Primer (antilysine)(10x diluted) before large scale culturing
 +
    brightest colony with 100ml LB AMP-CM and making glycerol stocks.
Results:
Results:
Line 93: Line 98:
Michelle:
Michelle:
-
     Digest LRP, SBP+LRP, and TET-RBS-SBP-LRP-TERM with EcoRI and PSTI, followed by ligation into PSBC13 (digested with EcoRI and PSTI),  and transformation onto LB-CM plates.  
+
     Digest LRP, SBP+LRP, and TET-RBS-SBP-LRP-TERM with EcoRI and PSTI
 +
    followed by ligation into PSBC13 (digested with EcoRI and PSTI)
 +
    transformation onto LB-CM plates.  
Results:
Results:

Latest revision as of 01:44, 27 October 2012



Protocols | Week 1 | Week 2 | Week 3 | Week 4 | Week 5 | Week 6 | Week 7 | Week 8 | Week 9 | Week 10 | Week 11 | Week 12 | Week 13 | Week 14 | Week 15 | Week 16 | Week 17 | Week 18 | Week 19 | Final Weeks |


Week 18: September 17 - September 21

Contents

September 17

Joe:
purify protein from 10, 10ml tubes
incubate with rice for 5 hours

September 18

Joe: RFP-SBP ligation for submission 

   culture more RFP-SBP in Top10 cells
   Successful sticky of protein to rice, got pictures

Michelle:

Small cultured two successful colonies from M2-BL21 transformation with LB AMP-CM.


Justin and Dafne:
Express more SBP-B12 protein
Using 96-well binding plate, add 100 ul of concentrated protein to 10 wells
incubate overnight

September 19

Joe: Transform RFP-SBP for submission

Justin and Dafne 

   Rinse plate 5x using PBS-T with 5% non-fat milk
   Add 100 ul of blocking buffer (PBS-T with 5% non-fat milk) to 10 wells with protein as well as 10 wells with no protein (control)
   Incubate overnight
   Purify yesterdays protein expression using amylose column (as done before)
   Digest SBP, B12, SBP-B12, SBP-B12 in expression plasmid by EcoRI HF and Pst I HF


September 20

Joe: Culture more RFP 

   Digest SBP by EcoRI/PstI for submission
   RFP-SBP: make 10 tubes from glycerol stock
   Colony PCR of RFP-SBP for submission

Michelle: 

   PCR colony check M2-BL21 
   Forward Primer (SBP-sense124)(10x diluted) and Reverse Primer (antilysine)(10x diluted) before large scale culturing  
   brightest colony with 100ml LB AMP-CM and making glycerol stocks.

Results: PCR colony was very successful showed that both colonies in lanes #2 and #3 had bright bands [Gel #198]. Therefore indicating large scale culturing could be proceeded.

Justin and Dafne 

   Add 10 ul of vitamin B12-HRP to 1 ml of PBS-T
   Make 4 more 100 fold dilutions of above solution
   Add each diluted solution to 2 wells
   incubate overnight
   Run Western blot analysis and Coomassie stain analysis of purified protein from yesterday
   Ligate yesterday’s digestions into submission plasmid, transform into TOP10 cells

Jeremiah & Chris: · Express the TBP+ gene using an L. Arabinose gradient · Digest TBP, SBP, TBP+, TBP+++, and LA-TBP+ o Run on gel (gel #200) · Ligate the genes above into pSB1C3 · Make glycerol stock of LA-TBP+ in BL21 cells

September 21

Michelle: 

   Digest LRP, SBP+LRP, and TET-RBS-SBP-LRP-TERM with EcoRI and PSTI
   followed by ligation into PSBC13 (digested with EcoRI and PSTI)
   transformation onto LB-CM plates.

Results: LRP, SBP+LRP, and TET-RBS-SBP-LRP-TERM into PSBC13 for iGEM submission.

Justin and Dafne 

   Rinse wells with PBS-T 5x
   Rinse well with PBS 3x
   Add TMB-Microwell peroxidase to each well
   Digest plasmids again


Jeremiah & Chris: · Purify pSB1C3 again via glass milk purification o Run on gel (gel #203) · Dephosphorylate pSB1C3

LA-TBP+ PCR check

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