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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Open Chromatin</span>
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Open Chromatin</span>
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
|style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}}
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'''LCR Assembly'''
'''LCR Assembly'''


'''Prep the Bridge Oligos (BO)'''<br>
# MV10/Gal4DB-mCh bridge: LCRb_MV10tctGal4DB_rc
# Gal4DB-mCh/ATF2 bridge: LCRb_mCh_ATF2_rc
# ATF2/MV10 bridge: LCRb_ATF2tcaMV10_rc
* 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)
{| {{table}}
|-
| Reagent || BO 1 || BO 2 || BO 3
|-
| 100 μM Stock BO || 3.0 || 3.0 || 3.0
|-
| dH<sub>2</sub>O || 97.0 || 97.0 || 97.0
|-
| &nbsp; || 100.0 || 100.0 || 100.0
|}
'''Prep the dsDNA fragments'''<br>
# MV10 (XbaI-cut and mung bean-blunted)
# Gal4DB-mCherry (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>
** 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
Table: Dilute each purified dsDNA to 30 fmol/μL (30 nM) in a final volume of 50 μL (in 1.5 mL tubes)
{| {{table}}
|-
| Reagent || DNA 1 || DNA 2 || DNA 3
|-
| DNA || x μL || x μL || x μL
|-
| dH<sub>2</sub>O || ## || ## || ##
|-
| &nbsp; || 50.0 || 50.0 || 50.0
|}
'''PNK treatments'''
# MV10 + Gal4DB-mCh + ATF2
# MV10 (neg ctrl.)


'''PNK treatment'''
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
* 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'''
Table: Set up the following in PCR tubes:
{| {{table}}
|-
| Reagent || PNK 1 || PNK 2
|-
| 30 fmol/μL MV10 || 2.0 || 2.0
|-
| 30 fmol/μL Gal4DB-mCh || 2.0 || ---
|-
| 30 fmol/μL ATF2 || 2.0 || ---
|-
| 10x T4 Ligation buf (NEB) || 1.0 || 1.0
|-
| T4 PNK (NEB) || 0.5 || 0.5
|-
| dH<sub>2</sub>O || 2.5 || 6.5
|-
| &nbsp; || 10.0 μL || 10.0 μL
|}


Procedure:
* Thermal cycler: program the following...
* Dilute the purified dsDNA to 30 fmol/μL (30 nM)
** Incubate at 37°C/ 30 min.
** 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.
** Heat-inactivate PNK at 65°C/ 20 min.
** 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
* Mix the appropriate amount of dsDNA fragments together and treat with Polynucleotide kinase to add 5'-phosphates (see table below)




PNK reaction
'''LCR Reactions'''
# MV10 + Gal4DB-mCh + ATF2
# MV10 (neg. ctrl.)
 
* 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 multiplier = number of LCR reactions + 1 (for pipetting error)
 
Master Mix (in 1.5 mL tube)
{| {{table}}
{| {{table}}
| Reagent || (Single Rxn.) || MM x3
|-
| Oligo Bridge 1 || (2.0) || 6.0
|-
|-
| Reagent || Volume
| Oligo Bridge 2 || (2.0) || 6.0
|-
|-
| 30 fmol/μL MV10 || 2.0
| Oligo Bridge 3 || (2.0) || 6.0
|-
|-
| 30 fmol/μL Gal4DB-mCh || 2.0
| 10X Ampligase Buffer || (2.0) || 6.0
|-
|-
| 30 fmol/μL ATF2 || 2.0
| Ampligase || (1.0) || 1.0
|-
| dH<sub>2</sub>O || (1.0) || 3.0
|-
|-
| 10x T4 Ligation buf (NEB) || 1.0
| &nbsp; || (10.0) || 30.0
|}
 
Table: Add master mix directly into 10 μL PNK DNA tubes
{| {{table}}
|-
|-
| T4 PNK (NEB) || 0.5
| Reagent || LCR 1 || LCR 2
|-
|-
| dH<sub>2</sub>O || 2.5 μL
| PNK DNA || 10.0 || 10.0
|-
|-
| &nbsp; || 10.0 μL
| Master Mix || 10.0 || 10.0
|-
| &nbsp; || 20.0 || 20.0
|}
|}


* Incubate at 37°C/ 30 min.
* Run LCR...
* Heat-inactivate PNK at 65°C/ 20 min.
** Look for "LCR" program on one of the PCR machines
Program: LCR
* 94°C, 2 min
* 50x[94°C, 10 sec; 55°C, 30 sec; 66°C, 60 sec]
* 4°C ∞
 


Transformation
* Carry out the usual transformation, Quick Method: http://openwetware.org/wiki/Haynes:TransformationPlasmids




Latest revision as of 01:04, 27 September 2017

Open Chromatin Main project page
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ASSEMBLY of MV10, ATF2, and Gal4DB/mCherry

  • LCR Assembly


LCR Assembly

Prep the Bridge Oligos (BO)

  1. MV10/Gal4DB-mCh bridge: LCRb_MV10tctGal4DB_rc
  2. Gal4DB-mCh/ATF2 bridge: LCRb_mCh_ATF2_rc
  3. ATF2/MV10 bridge: LCRb_ATF2tcaMV10_rc
  • 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)

Reagent BO 1 BO 2 BO 3
100 μM Stock BO 3.0 3.0 3.0
dH2O 97.0 97.0 97.0
  100.0 100.0 100.0


Prep the dsDNA fragments

  1. MV10 (XbaI-cut and mung bean-blunted)
  2. Gal4DB-mCherry (Phusion PCR)
  3. 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.
    • 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


Table: Dilute each purified dsDNA to 30 fmol/μL (30 nM) in a final volume of 50 μL (in 1.5 mL tubes)

Reagent DNA 1 DNA 2 DNA 3
DNA x μL x μL x μL
dH2O ## ## ##
  50.0 50.0 50.0


PNK treatments

  1. MV10 + Gal4DB-mCh + ATF2
  2. MV10 (neg ctrl.)

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
  • 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

Table: Set up the following in PCR tubes:

Reagent PNK 1 PNK 2
30 fmol/μL MV10 2.0 2.0
30 fmol/μL Gal4DB-mCh 2.0 ---
30 fmol/μL ATF2 2.0 ---
10x T4 Ligation buf (NEB) 1.0 1.0
T4 PNK (NEB) 0.5 0.5
dH2O 2.5 6.5
  10.0 μL 10.0 μL
  • Thermal cycler: program the following...
    • Incubate at 37°C/ 30 min.
    • Heat-inactivate PNK at 65°C/ 20 min.


LCR Reactions

  1. MV10 + Gal4DB-mCh + ATF2
  2. MV10 (neg. ctrl.)
  • 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 multiplier = number of LCR reactions + 1 (for pipetting error)

Master Mix (in 1.5 mL tube)

Reagent (Single Rxn.) MM x3
Oligo Bridge 1 (2.0) 6.0
Oligo Bridge 2 (2.0) 6.0
Oligo Bridge 3 (2.0) 6.0
10X Ampligase Buffer (2.0) 6.0
Ampligase (1.0) 1.0
dH2O (1.0) 3.0
  (10.0) 30.0

Table: Add master mix directly into 10 μL PNK DNA tubes

Reagent LCR 1 LCR 2
PNK DNA 10.0 10.0
Master Mix 10.0 10.0
  20.0 20.0
  • Run LCR...
    • Look for "LCR" program on one of the PCR machines

Program: LCR

  • 94°C, 2 min
  • 50x[94°C, 10 sec; 55°C, 30 sec; 66°C, 60 sec]
  • 4°C ∞


Transformation



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