User:Karmella Haynes/Notebook/BioBrick cloning/2015/04/10: Difference between revisions

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* LCR development - Backbone PCR, Phusion and temp gradient, re-do (do this on Monday)
* LCR development - Backbone PCR, Phusion and temp gradient, re-do (do this on Monday)
----
'''LCR development - Backbone PCR'''<br>
* Repeat PCR from 04/09/15, use fresh dNTP's
* '''MV10''' (5191 bp): Template - MV10 plasmid, F - MV10 f1, R - MV10 r1
* Gradient PCR to enhance 5 kb band
# 68°C
# 67.3°C
# 65.9°C
# 63.9°C
# 61.4°C
# 59.6°C
# 58.1°C
# 57°C
{| {{table}} cellspacing="3" <!-- PCR rxn. table -->
|- valign="top"
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Rxn1-8
| bgcolor=#cfcfcf | (x9)
| rowspan="7" | Expected:<br>1. Gal4DB-mCh = 1170<br>3,4. ATF2 = 906<br>5,6. MV10 = 5191
| rowspan="7" | [[Image:somegel.jpg|200px|Hover name]]<br>10 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
|-
| Template || 0.2 || 1.8
|-
| 10 uM fwd primer || 1.0 || 9.0
|-
| 10 uM rev primer || 1.0 || 9.0
|-
| 10 mM dNTPs || 1.0 || 9.0
|-
| Phusion pol. || 0.5 || 4.5
|-
| 5x HF buffer || 5.0 || 45.0
|-
| dH<sub>2</sub>O || 41.3 || 371.7
|-
| &nbsp; || 50.0
|}
Program: Phusion (block A) - edited to include 65°C - 55°C annealing gradient
* 98°C, 3 min
* 30x[98°C, 10 sec; 68°C - 57°C, 30 sec; 72°C, 2.5 min]
* 72°C, 10 min
* 4°C ∞
Conclusions
* tba




Revision as of 13:20, 16 April 2015

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04/10/15

  • LCR development - Backbone PCR, Phusion and temp gradient, re-do (do this on Monday)




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