User:Daniel A. Vargas/Notebook/General lab notebook/2015/08/04: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
Line 16: Line 16:


Prep the Bridge Oligos<br>
Prep the Bridge Oligos<br>
Note: Final conc. in LCR rxn. is 30 nM each
Note: Final conc. in LCR rxn. will end up being 30 nM each
# 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''
# Use 3.0 μL of oligo working sln. per 30 μL LCR reaction
# Use 3.0 μL of oligo working sln. per 30 μL LCR reaction
Line 27: Line 27:
** Formula: x μL = length in bp ÷ measured ng/μL *  ''0.0195 ng/μL'' * 50μL
** Formula: x μL = length in bp ÷ measured ng/μL *  ''0.0195 ng/μL'' * 50μL
* Use 2.0 μL of '''each''' diluted dsDNA per 10 μL PNK reaction
* Use 2.0 μL of '''each''' diluted dsDNA per 10 μL PNK reaction
* Mix the appropriate amount of dsDNA fragments together and treat with Polynucleotide kinase to add 5'-phosphates (see table below)
* Treat the mixed dsDNAs with Polynucleotide Kinase (PNK) to add 5'-phosphates (see table below)


Set up the following in PCR tubes:
{| {{table}}
{| {{table}}
|-
|-
Line 48: Line 49:
|}
|}


* Incubate at 37°C/ 30 min.
* Thermal cycler: program the following...
* Heat-inactivate PNK at 65°C/ 20 min.
** Incubate at 37°C/ 30 min.
** Heat-inactivate PNK at 65°C/ 20 min.




LCR Reaction
LCR Reaction
* Add components to the PNK DNA reaction 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
* Set up the reactions in PCR-sized tubes
* Best way to do this is to make a master mix, then aliquot into PNK DNA


Master Mix (in 1.5 mL tube)
{| {{table}}
{| {{table}}
| Reagent || Volume
| Reagent || Volume
|-
|-
| PNK DNA || 10.0 μL
| Oligo Bridge 1 || 2.0 (each)
|-
| Oligo Bridge || 3.0 (each)
|-
|-
| 10X Ampligase Buffer || 3.0
| 10X Ampligase Buffer || 2.0
|-
|-
| Ampligase || 1.0
| Ampligase || 1.0
Line 71: Line 72:
| &nbsp; || 20.0 μL
| &nbsp; || 20.0 μL
|}
|}
Add master mix into 10 μL PNK DNA
{|
|-
| Reagent || Volume
|-
| PNK DNA || 10.0 μL


* Run LCR...
* Run LCR...

Revision as of 07:03, 4 August 2015

Open Chromatin <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

ASSEMBLY of MV10, ATF2, and Gal4DB/mCherry

  • LCR Assembly


LCR Assembly

Prep the Bridge Oligos
Note: Final conc. in LCR rxn. will end up being 30 nM each

  1. Make a 300 nM working solution (final volume = 100 μL) in a new tube. 3 μL of 100μM oligo stock + 97 μL dH2O = 100 μL
  2. Use 3.0 μL of oligo working sln. per 30 μL LCR reaction


PNK treatment
Overview: Mix double-stranded DNA fragments, treat with PNK to add phosphate groups

  • Dilute the purified dsDNA to 30 fmol/μL (30 nM)
    • The volume of purified DNA (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
  • Use 2.0 μL of each diluted dsDNA per 10 μL PNK reaction
  • Treat the mixed dsDNAs with Polynucleotide Kinase (PNK) to add 5'-phosphates (see table below)

Set up the following in PCR tubes:

Reagent Volume
30 fmol/μL MV10 2.0
30 fmol/μL Gal4DB-mCh 2.0
30 fmol/μL ATF2 2.0
10x T4 Ligation buf (NEB) 1.0
T4 PNK (NEB) 0.5
dH2O 2.5 μL
  10.0 μL
  • Thermal cycler: program the following...
    • Incubate at 37°C/ 30 min.
    • Heat-inactivate PNK at 65°C/ 20 min.


LCR Reaction

  • 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

Master Mix (in 1.5 mL tube)

Reagent Volume
Oligo Bridge 1 2.0 (each)
10X Ampligase Buffer 2.0
Ampligase 1.0
dH2O 3.0 μL
  20.0 μL

Add master mix into 10 μL PNK DNA

Reagent Volume
PNK DNA 10.0 μL
  • Run LCR...
    • Look for "LCR" program on one of the PCR machines


Transformation

  • Carry out the usual transformation. (quick and dirty.)
Retrieved from "https://openwetware.org/mediawiki/index.php?title=User:Daniel_A._Vargas/Notebook/General_lab_notebook/2015/08/04&oldid=895788"