User:Daniel A. Vargas/Notebook/General lab notebook/2015/08/04: Difference between revisions
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'''Prep the dsDNA fragments''' | '''Prep the dsDNA fragments''' | ||
# MV10 (XbaI-cut and mung bean-blunted) | # MV10 (XbaI-cut and mung bean-blunted) | ||
# Gal4DB-mCherry | # Gal4DB-mCherry (Phusion PCR) | ||
# ATF2 | # ATF2 (Phusion PCR) | ||
* | * Calculations: 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> | ||
** MV10: length in bp ÷ measured ng/μL * ''0.0195 ng/μL'' * 50μL''' = x μL | |||
** Gal4DB-mCherry: length in bp ÷ measured ng/μL * ''0.0195 ng/μL'' * 50μL''' = x μL | |||
** ATF2: length in bp ÷ measured ng/μL * ''0.0195 ng/μL'' * 50μL''' = x μL | |||
* Dilute the purified dsDNA to 30 fmol/μL (30 nM) in a final volume of 50 μL | * Dilute the purified dsDNA to 30 fmol/μL (30 nM) in a final volume of 50 μL | ||
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| Reagent || DNA 1 || DNA 2 || DNA 3 | | Reagent || DNA 1 || DNA 2 || DNA 3 | ||
|- | |- | ||
| DNA || ### | | DNA || x μL || x μL || x μL | ||
|- | |||
| dH<sub>2</sub>O || ## || ## || ## | |||
|- | |||
| || 50.0 || 50.0 || 50.0 | |||
|} | |} | ||
Revision as of 13:22, 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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