User:Karmella Haynes/Notebook/BioBrick cloning/2015/04/17: Difference between revisions

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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Karmella's BioBrick Cloning</span>
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Karmella's BioBrick Cloning</span>
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
|style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}}
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==04/16/15==
==04/17/15==
<!-- Precede finished items with a checkmark &#x2713; -->
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* Rene - gRNA plasmids, 5 mL cultures (11 total)
* Rene - gRNA plasmids, 5 mL cultures (11 total)
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* Stage 1 - insert Regulator ORFS
* Stage 1 - insert Regulator ORFS
** Phusion PCR-amplify ORFs (add 5'-E, 3'-X cut sites)
** Phusion PCR-amplify ORFs (add 5'-E, 3'-X cut sites)
** Insert ORF(E/X) into Vector (#/#). see [https://benchling.com/s/VFNzuauP/edit Vector plasmid map]
** Insert ORF(E/X) into Vector (E/X). see [https://benchling.com/s/VFNzuauP/edit Vector plasmid map]
* Stage 2 - insert Promoters
* Stage 2 - insert Promoters
** Cut & dephos promoters with E/X
** Cut & dephos promoters with E/X
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| bgcolor=#cfcfcf | Volume  
| bgcolor=#cfcfcf | Volume  
| bgcolor=#cfcfcf | Mix (x5)
| bgcolor=#cfcfcf | Mix (x5)
| rowspan="7" | <u>Expected:</u><br>1. BB 1 = size<br>2. BB2 = size<br>
| rowspan="7" | <u>Expected:</u><br>1. AubR = 851<br>2. BjaR = 776<br>3. BraR = 776<br>4. RpaR = 779<br>
| rowspan="7" | <!-- [[Image:GelImage.jpg|400px|Hover name]]<br>15 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] -->
| rowspan="7" | [[Image:KAH041715_gel1.jpg|200px|Hover name]]<br>5 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
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| DNA(plasmid) || 0.5 μL || ---
| DNA(plasmid) || 0.5 μL || ---
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Program: Phusion (block B)
Program: Phusion (block B)
* 98°C, 3 min
* 98°C, 3 min
* 35x[98°C, 10 sec; 66°C, 30 sec; 72°C, ?? sec]
* 35x[98°C, 10 sec; 66°C, 30 sec; 72°C, 60 sec]
* 72°C, 10 min
* 72°C, 10 min
* 4°C ∞
* 4°C ∞


Conclusion
Conclusion
* tba
* All four worked. Proceed to next step




----
----
'''LCR Development'''
'''LCR Development'''
* Repeat Phusion for MV10, use correct amount of 5x buffer
* Repeat Phusion for MV10 (from 4/16/15), use correct amount of 5x buffer




Phusion PCR
Phusion PCR
# AubR, EcoRI F/ XbaI R
# BjaR, "
# BraR, "
# RpaR, "


{| {{table}} border="1" cellspacing="3" <!-- Phusion PCR table -->
{| {{table}} border="1" cellspacing="3" <!-- Phusion PCR table -->
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| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Rxn1,2
| bgcolor=#cfcfcf | Rxn1,2
| rowspan="7" | <u>Expected:</u><br>1. BB 1 = size<br>2. BB2 = size<br>
| rowspan="7" | <u>Expected:</u><br>1,2. MV10 = 5191
| rowspan="7" | <!-- [[Image:GelImage.jpg|400px|Hover name]]<br>15 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] -->
| rowspan="7" | [[Image:KAH041715_gel2.jpg|150px|Hover name]]<br>15 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
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| DNA(clean linear) || 1.0 μL
| DNA(clean linear) || 1.0 μL
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Program: Phusion (block #)
Program: Phusion (block A)
* 98°C, 3 min
* 98°C, 3 min
* 35x[98°C, 10 sec; 60°C, 30 sec; 72°C, 2.5 min]
* 35x[98°C, 10 sec; 60°C, 30 sec; 72°C, 2.5 min]
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Conclusion
Conclusion
* tba
* Big improvement. Keep this for potential cloning
* Also try again with higher annealing temp




Latest revision as of 00:55, 27 September 2017

Karmella's BioBrick Cloning Main project page
Previous entry      Next entry

04/17/15

  • Rene - gRNA plasmids, 5 mL cultures (11 total)
  • Ryan - Receiver plasmids, assembly
  • LCR Development - Repeat Phusion for MV10, use correct amount of 5x buffer


Ryan - Receiver plasmids, assembly

  • Stage 1 - insert Regulator ORFS
    • Phusion PCR-amplify ORFs (add 5'-E, 3'-X cut sites)
    • Insert ORF(E/X) into Vector (E/X). see Vector plasmid map
  • Stage 2 - insert Promoters
    • Cut & dephos promoters with E/X
    • Insert promoters(E/X) into Vector/Regulator (BbsI) via Lig/Dig cycling


Phusion PCR

  1. AubR, EcoRI F/ XbaI R
  2. BjaR, "
  3. BraR, "
  4. RpaR, "
Reagent Volume Mix (x5) Expected:
1. AubR = 851
2. BjaR = 776
3. BraR = 776
4. RpaR = 779
 Hover name
5 μL/lane; 1% agarose; Ladder
DNA(plasmid) 0.5 μL ---
10 μM F primer 1.0 5.0
10 μM R primer 1.0 5.0
10 mM dNTPs 1.0 5.0
Phusion Pol. 0.5 2.5
5X HF buffer 10.0 50.0
dH2O 36.0 180.0
  50.0


Program: Phusion (block B)

  • 98°C, 3 min
  • 35x[98°C, 10 sec; 66°C, 30 sec; 72°C, 60 sec]
  • 72°C, 10 min
  • 4°C ∞

Conclusion

  • All four worked. Proceed to next step


LCR Development

  • Repeat Phusion for MV10 (from 4/16/15), use correct amount of 5x buffer


Phusion PCR

Reagent Rxn1,2 Expected:
1,2. MV10 = 5191 		Hover name
15 μL/lane; 1% agarose; Ladder
DNA(clean linear) 1.0 μL
10 μM F primer 1.0
10 μM R primer 1.0
10 mM dNTPs 1.0
Phusion Pol. 0.5
5X HF buffer 10.0
dH2O 35.5
  50.0


Program: Phusion (block A)

  • 98°C, 3 min
  • 35x[98°C, 10 sec; 60°C, 30 sec; 72°C, 2.5 min]
  • 72°C, 10 min
  • 4°C ∞

Conclusion

  • Big improvement. Keep this for potential cloning
  • Also try again with higher annealing temp



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