User:Tara K. Luckau/Notebook/Team ConGen/Entry Base: Difference between revisions
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# Scun2-R-pigtail | # Scun2-R-pigtail | ||
===Calculations=== | ===Calculations=== | ||
* | * Scun2-F | ||
** 28.3nmol + 141.5 Low TE = 200µM STK | |||
* Scun2-R | |||
** 29.2nmol + 146µL Low TE = 200µM STK | |||
* Scun2-F-M13F(-21) | |||
** 26.6nmol + 133µL Low TE = 200µM STK | |||
* Scun2-R-pigtail | |||
** 30.5nmol + 152.5µL Low TE = 200µM STK | |||
* stored overnight in fridge to rehydrate completely before making dilution to 20µM | |||
==18 October 2010 - dNTP Mix== | |||
===Purpose=== | |||
* Mix dNTPs and aliquot for PCR | |||
===Calculations=== | |||
* Have: Invitrogen 100mM dNTP Set (catalog# 10297-018) (250µL of 25µmol in H2O @ 100mM each) | |||
* Want: 10mM dNTP mix (2.5mM each), 5 aliquots of 1000µL | |||
* (C1)(V1)=(C2)(V2) | |||
*: (2.5mM)(5000µL)=(100mM)(V2) | |||
*: V2=125µL of each dNTP | |||
===Wet Work=== | |||
* 125µL dATP + 125µL dTTP + 125µL dCTP + 125µL dGTP + 4500µL H2O = 5000µL dNTP Mix @ 10mM (2.5mM each dNTP) | |||
* Used NanoPure H20 from room 325 | |||
* Aliquots: 1000µL into each of 5 snap-caps; 4 stored in freezer, 1 stored in fridge for immediate use | |||
==19 October 2010 - PCR Scun2 F-R== | |||
[[Image:Luckau_PCR20101019.jpg]] | |||
* DNA = SCOC CPN 82 | |||
* 2 mats with lab tape, no evaporation! | |||
==21 October 2010 - Gel Scun2 F-R== | |||
===Make 1x TAE=== | |||
* 20mL 50x TAE + 980 mL NanoPure H2O | |||
===Cast Gel=== | |||
* 100mL 1x TAE + 1.5g agarose | |||
* large stirbar, hotplate set at 150 for about 30 min | |||
* let cool about 20 min | |||
* not enough volume! FAIL! | |||
===Next Try=== | |||
* 270mL TAE + 4.05g agarose | |||
* (for future reference, total volume ≈ volume of gel box in cm<sup>3</sup>, ie 27cm long x 20 cm wide x 0.5 cm thick gel = 270mL of TAE) | |||
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Revision as of 12:31, 25 October 2010
Tara's Lab Notebook | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page |
27 September 2010 - Ordered Primers
11 October 2010 - PCR Thermalcycler TestingPurpose
Results
Purpose
Results
12 October 2010 - PCR Thermalcycler TestingPurpose
Results
18 October 2010 - PCR Thermalcycler TestingPurpose
Results
==18 October 2010 - Primer Rehydration, Scun2 Purpose
Calculations
18 October 2010 - dNTP MixPurpose
Calculations
Wet Work
19 October 2010 - PCR Scun2 F-R
21 October 2010 - Gel Scun2 F-RMake 1x TAE
Cast Gel
Next Try
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