User:Karmella Haynes/Notebook/BioBrick cloning/2015/04/16: Difference between revisions

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| 1. MV10 XbaI || 0.017 || 1.47 || 17.24
| 1. MV10 XbaI || 0.017 || 1.47 || 17.24
|}
|}
Phusion PCR
# 1.0 μL template, HF buffer
# 2.0 μL template, HF buffer
# 1.0 μL template, GC buffer
# 2.0 μL template, GC buffer
{| {{table}} cellspacing="3" <!-- PCR rxn. table -->
|- valign="top"
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Rxn1,2
| bgcolor=#cfcfcf | Rxn3,4
| rowspan="7" | Expected:<br>1,2. Gal4DB-mCh = 1170<br>3,4. ATF2 = 906<br>
| 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 || 1.0 || 2.0
|-
| 10 uM fwd primer || 1.0 || 1.0
|-
| 10 uM rev primer || 1.0 || 1.0
|-
| 10 mM dNTPs || 1.0 || 1.0
|-
| Phusion pol. || 0.5 || 0.5
|-
| 5x buffer (HF/GC) || 5.0 || 5.0
|-
| dH<sub>2</sub>O || 40.5 || 39.5
|-
| &nbsp; || 50.0 || 50.0
|}
Program: Phusion (block B)
* 98°C, 3 min
* 35x[98°C, 10 sec; 60°C, 30 sec; 72°C, 30 sec]
* 72°C, 10 min
* 4°C ∞




Revision as of 11:57, 16 April 2015

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04/16/15

  • LCR Development - PCR MV10 bacbone


LCR Development

  • Try PCR on linearized vector
Reagent Volume
DNA(plasmid) 5.0 μL
10X buffer 3.0
XbaI 2.0
dH2O 20.0
  30 μL --> 37°C/ ~30 min.


Measure conc.

  • DNA purified with Sigma PCR clean-up kit
  • Eluted& back-eluted with 30 μL elution sln.
Sample OD260 260/280 ng/μL
1. MV10 XbaI 0.017 1.47 17.24


Phusion PCR

  1. 1.0 μL template, HF buffer
  2. 2.0 μL template, HF buffer
  3. 1.0 μL template, GC buffer
  4. 2.0 μL template, GC buffer
Reagent Rxn1,2 Rxn3,4 Expected:
1,2. Gal4DB-mCh = 1170
3,4. ATF2 = 906
 Hover name
10 μL/lane, 1% agarose; Ladder
Template 1.0 2.0
10 uM fwd primer 1.0 1.0
10 uM rev primer 1.0 1.0
10 mM dNTPs 1.0 1.0
Phusion pol. 0.5 0.5
5x buffer (HF/GC) 5.0 5.0
dH2O 40.5 39.5
  50.0 50.0

Program: Phusion (block B)

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




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