User:Tara K. Luckau/Notebook/Team ConGen/2011/01/14: Difference between revisions

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Line 35: Line 35:
| || 10January2011 (fail) || 13January2011 (worked)
| || 10January2011 (fail) || 13January2011 (worked)
|-
|-
| Buffer / MgCl<sub>2</sub> || 1.5 || 1
| Buffer / MgCl<sub>2</sub> || 1.5 µL || 1 µL
|-
|-
| Primers || 0.1 each || 0.25 each
| Primers || 0.1 µL each || 0.25 µL each
|-style="background: yellow"
|-style="background: yellow"
| Taq || 0.05 (0.25 Units/reaction)|| 0.5 (2.5 Units/reaction)
| Taq || 0.05 µL (0.25 Units)|| 0.5 µL (2.5 Units)
|}
|}


The largest difference between the two reactions was Taq - 10-fold difference
The largest difference between the two reactions was Taq - 10-fold difference


In the interest of cost, try to pull this down to 1 Unit/reaction (0.2µL)
* In the interest of cost, try to pull this down to 1 Unit/reaction (0.2µL)
* Also re-calculate for 10µL reaction volume
* Find annealing temperatures (touchdowns are poopy)
 
 
 
==PCR decrease Taq==
* [[Image:20110114_PCROpt_Touchdown.jpg|900 px]]




Revision as of 16:24, 14 January 2011

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Gel Lance conditions

Success!!!



  • Scun2 Primer - expecting bands ~150bp
    • Scun sample - band at 130bp
    • Scoc sample - band at 75bp


  • Scun22 Primer - expecting bands at ~450bp
    • Scun sample - multiple bands; but there is one prominent band in one of the replicates at 450bp
    • Scoc sample - multiple bands; but prominent band in all three replicates at 450bp


Differences between Lance conditions and previous trials

10January2011 (fail) 13January2011 (worked)
Buffer / MgCl2 1.5 µL 1 µL
Primers 0.1 µL each 0.25 µL each
Taq 0.05 µL (0.25 Units) 0.5 µL (2.5 Units)

The largest difference between the two reactions was Taq - 10-fold difference

  • In the interest of cost, try to pull this down to 1 Unit/reaction (0.2µL)
  • Also re-calculate for 10µL reaction volume
  • Find annealing temperatures (touchdowns are poopy)


PCR decrease Taq
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