User:Daniel A. Vargas/Notebook/General lab notebook/2015/08/04: Difference between revisions
From OpenWetWare
(fix raw html notebook nav) |
|||
(5 intermediate revisions by 2 users not shown) | |||
Line 2: | Line 2: | ||
|- | |- | ||
|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"| | |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]] }}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}} | ||
|- | |- | ||
| colspan="2"| | | colspan="2"| | ||
Line 16: | Line 16: | ||
'''Prep the Bridge Oligos (BO)'''<br> | '''Prep the Bridge Oligos (BO)'''<br> | ||
# MV10/Gal4DB bridge: LCRb_MV10tctGal4DB_rc | # 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. | * 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. | ||
Line 71: | Line 71: | ||
{| {{table}} | {| {{table}} | ||
|- | |- | ||
| Reagent || | | Reagent || PNK 1 || PNK 2 | ||
|- | |- | ||
| 30 fmol/μL MV10 || 2.0 || 2.0 | | 30 fmol/μL MV10 || 2.0 || 2.0 | ||
Line 99: | Line 99: | ||
* 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 multiplier = number of LCR reactions + 1 (for pipetting error) | ||
Master Mix (in 1.5 mL tube) | Master Mix (in 1.5 mL tube) | ||
{| {{table}} | {| {{table}} | ||
| Reagent || | | Reagent || (Single Rxn.) || MM x3 | ||
|- | |- | ||
| Oligo Bridge 1 || 2.0 || 6.0 | | Oligo Bridge 1 || (2.0) || 6.0 | ||
|- | |- | ||
| Oligo Bridge 2 || 2.0 || 6.0 | | Oligo Bridge 2 || (2.0) || 6.0 | ||
|- | |- | ||
| Oligo Bridge 3 || 2.0 || 6.0 | | Oligo Bridge 3 || (2.0) || 6.0 | ||
|- | |- | ||
| 10X Ampligase Buffer || 2.0 || 6.0 | | 10X Ampligase Buffer || (2.0) || 6.0 | ||
|- | |- | ||
| Ampligase || 1.0 || 1.0 | | Ampligase || (1.0) || 1.0 | ||
|- | |- | ||
| dH<sub>2</sub>O || | | dH<sub>2</sub>O || (1.0) || 3.0 | ||
|- | |- | ||
| || | | || (10.0) || 30.0 | ||
|} | |} | ||
Add master mix into 10 μL PNK DNA | Table: Add master mix directly into 10 μL PNK DNA tubes | ||
{| {{table}} | |||
|- | |||
| Reagent || LCR 1 || LCR 2 | |||
|- | |||
| PNK DNA || 10.0 || 10.0 | |||
|- | |- | ||
| | | Master Mix || 10.0 || 10.0 | ||
|- | |- | ||
| | | || 20.0 || 20.0 | ||
|} | |} | ||
* Run LCR... | * Run LCR... | ||
** Look for "LCR" program on one of the PCR machines | ** 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 | Transformation | ||
* Carry out the usual 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 Previous entry Next entry | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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)
Table: Add master mix directly into 10 μL PNK DNA tubes
Program: LCR
|