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

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----
----
'''Ryan - Receiver plasmids, assembly'''<br>
'''Ryan - Receiver plasmids, assembly'''
* 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)
Line 63: Line 63:
Conclusion
Conclusion
* tba
* tba
----
'''LCR Development'''
* Repeat Phusion for MV10, use correct amount of 5x buffer
Phusion PCR
# AubR, EcoRI F/ XbaI R
# BjaR, "
# BraR, "
# RpaR, "
{| {{table}} border="1" cellspacing="3" <!-- Phusion PCR table -->
|- valign="top"
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Rxn1,2
| rowspan="7" | <u>Expected:</u><br>1. BB 1 = size<br>2. BB2 = size<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] -->
|-
| 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
|-
| dH<sub>2</sub>O || 35.5
|-
| &nbsp; || 50.0 ||
|}
Program: Phusion (block #)
* 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
* tba




Revision as of 12:11, 17 April 2015

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04/16/15

  • 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 (#/#). 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. BB 1 = size
2. BB2 = size
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, ?? sec]
  • 72°C, 10 min
  • 4°C ∞

Conclusion

  • tba


LCR Development

  • Repeat Phusion for MV10, use correct amount of 5x buffer


Phusion PCR

  1. AubR, EcoRI F/ XbaI R
  2. BjaR, "
  3. BraR, "
  4. RpaR, "
Reagent Rxn1,2 Expected:
1. BB 1 = size
2. BB2 = size
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 #)

  • 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

  • tba



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