User:Daniel A. Vargas/Notebook/General lab notebook/2015/08/04: Difference between revisions
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'''Prep the Bridge Oligos (BO)'''<br> | '''Prep the Bridge Oligos (BO)'''<br> | ||
# MV10/Gal4DB bridge: LCRb_MV10tctGal4DB_rc | # MV10/Gal4DB bridge: LCRb_MV10tctGal4DB_rc | ||
# | # | ||
# | # | ||
Make a 300 nM working solution (final volume = 100 μL) in a fresh tube for each Bridge Oligo (BO) | * Overview - Bridge Oligos need to be brought to a working concentration of 300 nanomolar (nM). Eventually, final conc. in LCR rxn. will end up being 30 nanomolar (nM) each. | ||
Table: Make a 300 nM working solution (final volume = 100 μL) in a fresh tube for each Bridge Oligo (BO) (in 1.5 mL tubes) | |||
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'''Prep the dsDNA fragments'''<br> | '''Prep the dsDNA fragments'''<br> | ||
# MV10 (XbaI-cut and mung bean-blunted) | # MV10 (XbaI-cut and mung bean-blunted) | ||
# Gal4DB-mCherry (Phusion PCR) | # Gal4DB-mCherry (Phusion PCR) | ||
# ATF2 (Phusion PCR) | # ATF2 (Phusion PCR) | ||
* Overview - dsDNA will be diluted to a concentration of 30 fmol/μL | |||
* 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> | * 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 | ** MV10: length in bp ÷ measured ng/μL * ''0.0195 ng/μL'' * 50μL''' = x μL | ||
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** ATF2: 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 | ||
Table: Dilute each purified dsDNA to 30 fmol/μL (30 nM) in a final volume of 50 μL (in 1.5 mL tubes) | |||
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* 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''' | * 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''' | ||
Set up the following in PCR tubes: | Table: Set up the following in PCR tubes: | ||
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Revision as of 13:33, 4 August 2015
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ASSEMBLY of MV10, ATF2, and Gal4DB/mCherry
LCR Assembly Prep the Bridge Oligos (BO)
Table: Make a 300 nM working solution (final volume = 100 μL) in a fresh tube for each Bridge Oligo (BO) (in 1.5 mL tubes)
Overview: Mix double-stranded DNA fragments, treat with PNK to add phosphate groups
Table: 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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