User:Daniel A. Vargas/Notebook/General lab notebook/2015/08/04: Difference between revisions
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PNK | PNK treatments<br> | ||
# MV10 + Gal4DB-mCh + ATF2 | |||
# MV10 (neg ctrl.) | |||
Overview: Mix double-stranded DNA fragments, treat with PNK to add phosphate groups | Overview: Mix double-stranded DNA fragments, treat with PNK to add phosphate groups | ||
* 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 | ||
* 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. '''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: | Set up the following in PCR tubes: | ||
{| {{table}} | {| {{table}} | ||
|- | |- | ||
| Reagent || Volume | | Reagent || Volume || Rxn1 || Rxn2 | ||
|- | |- | ||
| 30 fmol/μL MV10 || 2.0 | | 30 fmol/μL MV10 || 2.0 || 2.0 | ||
|- | |- | ||
| 30 fmol/μL Gal4DB-mCh || 2.0 | | 30 fmol/μL Gal4DB-mCh || 2.0 || --- | ||
|- | |- | ||
| 30 fmol/μL ATF2 || 2.0 | | 30 fmol/μL ATF2 || 2.0 || --- | ||
|- | |- | ||
| 10x T4 Ligation buf (NEB) || 1.0 | | 10x T4 Ligation buf (NEB) || 1.0 || 1.0 | ||
|- | |- | ||
| T4 PNK (NEB) || 0.5 | | T4 PNK (NEB) || 0.5 || 0.5 | ||
|- | |- | ||
| dH<sub>2</sub>O || 2.5 | | dH<sub>2</sub>O || 2.5 || 6.5 | ||
|- | |- | ||
| || 10.0 μL | | || 10.0 μL | ||
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LCR | LCR Reactions | ||
# MV10 + Gal4DB-mCh + ATF2 | |||
# MV10 | |||
* Using the entire PNK DNA reaction, add other components (into the same PCR tubes) as described in the table below | * Using the entire PNK DNA reaction, add other components (into the same PCR tubes) as described in the table below | ||
* Best way to do this is to make a master mix, then aliquot into PNK DNA | * Best way to do this is to make a master mix, then aliquot into PNK DNA | ||
* Master mix multiplier = number of LCR reactions | |||
Master Mix (in 1.5 mL tube) | Master Mix (in 1.5 mL tube) | ||
{| {{table}} | {| {{table}} | ||
| Reagent || Volume | | Reagent || Volume || | ||
|- | |- | ||
| Oligo Bridge 1 || 2.0 (each) | | Oligo Bridge 1 || 2.0 (each) | ||
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{| | {| | ||
|- | |- | ||
| Reagent || Volume | | Reagent || Volume || Rxn1 | ||
|- | |- | ||
| PNK DNA || 10.0 μL | | PNK DNA || 10.0 μL |
Revision as of 07:10, 4 August 2015
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ASSEMBLY of MV10, ATF2, and Gal4DB/mCherry
LCR Assembly Prep the Bridge Oligos
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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