User:Daniel A. Vargas/Notebook/General lab notebook/2015/08/04: Difference between revisions
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* Make a 300 nM working solution (final volume = 100 μL) in a new tube. ''3 μL of 100μM oligo stock + 97 μL dH<sub>2</sub>O = 100 μL'' | * Make a 300 nM working solution (final volume = 100 μL) in a new tube. ''3 μL of 100μM oligo stock + 97 μL dH<sub>2</sub>O = 100 μL'' | ||
* Will use 2.0 μL of oligo working sln. per 20 μL LCR reaction | * Will use 2.0 μL of oligo working sln. per 20 μL LCR reaction | ||
'''Prep the dsDNA fragments''' | |||
# MV10 (XbaI-cut and mung bean-blunted) | |||
# Gal4DB-mCherry - Phusion PCR | |||
# ATF2 - Phusion PCR | |||
* Calculation: The volume of purified dsDNA (x) you will need to dilute in a final volume of 50 μL = length in bp ÷ measured ng/μL * ''30 fmol/μL * 650 fg/fmol dsDNA ÷ 1,000,000 fg/ng'' * 50 μL final volume. <br>'''Formula: x μL = length in bp ÷ measured ng/μL * ''0.0195 ng/μL'' * 50μL''' | |||
* Dilute the purified dsDNA to 30 fmol/μL (30 nM) in a final volume of 50 μL | |||
{| {{table}} | |||
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| Reagent || DNA 1 || DNA 2 || DNA 3 | |||
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| DNA || ### | |||
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Revision as of 13:18, 4 August 2015
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ASSEMBLY of MV10, ATF2, and Gal4DB/mCherry
LCR Assembly Prep the Bridge Oligos
Note: Final conc. in LCR rxn. will end up being 30 nanomolar (nM) each
PNK treatments
Overview: Mix double-stranded DNA fragments, treat with PNK to add phosphate groups
Set up the following in PCR tubes:
Master Mix (in 1.5 mL tube)
Add master mix into 10 μL PNK DNA
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