Haynes Lab:Notebook/Short Projects/2015/05/28: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
(Autocreate 2015/05/28 Entry for Haynes_Lab:Notebook/Short_Projects) |
(fix raw html notebook nav) |
||
| (3 intermediate revisions by one other user not shown) | |||
| Line 2: | Line 2: | ||
|- | |- | ||
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Haynes Lab - Short Projects</span> | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Haynes Lab - Short Projects</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"| | ||
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | <!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | ||
== | ==Ben Nyer - 28/05/2015== | ||
'''Cloning gRNAs into pSPgRNA for dCas9-HT binding to luc gene''' | |||
* Follow the "Oligo annealing and cloning into backbone vectors - NEW" protocol | |||
Phosphorylate and anneal oligo pairs | |||
# g025 T/B | |||
# g031 T/B | |||
# g034 T/B | |||
# g048 T/B | |||
{| {{table}} cellspacing="3" <!-- Oligo annealing table --> | |||
|- valign="top" | |||
| bgcolor=#cfcfcf | Reagent | |||
| bgcolor=#cfcfcf | Rxn | |||
| bgcolor=#cfcfcf | Mix (5x) | |||
|- | |||
| 100 μM Oligo 1 || 1.0 || --- | |||
|- | |||
| 100 μM Oligo 2 || 1.0 || --- | |||
|- | |||
| 10x T4 Lign buf (NEB) || 1.0 || 5.0 | |||
|- | |||
| T4 PNK (NEB) || 0.5 || 2.5 | |||
|- | |||
| dH<sub>2</sub>O || 6.5 || 32.5 | |||
|- | |||
| || 10.0 | |||
|} | |||
Bio-Rad C1000 Touch Thermocycler: Olig PNK-Ann | |||
* 37°C, 30 min | |||
* 95°C, 5 min | |||
* Ramp down to 25°C, 5°C/1 min. [90/1 min, 85/1 min, 80/1 min, ... 25°C/1 min] | |||
* 25°C, ∞ | |||
Dilute the product(s) 1:250 | |||
* Add 2 μL product to 498 μL dH<sub>2</sub>O | |||
Digestion/ Ligation Reactions (Vector / dsOligo Insert) | |||
# pSPgRNA / g025 | |||
# pSPgRNA / g031 | |||
# pSPgRNA / g034 | |||
# pSPgRNA / g048 | |||
{| {{table}} cellspacing="3" <!-- Oligo annealing table --> | |||
|- valign="top" | |||
| bgcolor=#cfcfcf | Reagent | |||
| bgcolor=#cfcfcf | Rxn | |||
| bgcolor=#cfcfcf | Mix (x5) | |||
|- | |||
| 100 ng Vector || 0.5 || 2.5 | |||
|- | |||
| 1:250 dsOligo || 2.0 || --- | |||
|- | |||
| 10x FD buf || 2.0 || 10.0 | |||
|- | |||
| 10 mM DTT || 1.0 || 5.0 | |||
|- | |||
| 10 mM ATP || 1.0 || 5.0 | |||
|- | |||
| FastDigest BbsI || 1.0 || 5.0 | |||
|- | |||
| T4 DNA ligase || 0.5 || 2.5 | |||
|- | |||
| dH<sub>2</sub>O || 12.0 || 60.0 | |||
|- | |||
| || 20.0 | |||
|} | |||
Bio-Rad C1000 Touch Thermocycler: BbsI DigLig | |||
* 6x [37°C, 5 min; 23°C, 5 min] | |||
* 4°C, ∞ | |||
Transformation(s) | |||
* Split ligations in 1/2, do rapid protocol for 1 set, long protocol for other set if needed | |||
* 10.0 μL ligation + 50 μL DH5α-turbo | |||
* Plate on 100 μg/mL amp | |||
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### --> | <!-- ##### DO NOT edit below this line unless you know what you are doing. ##### --> | ||
Latest revision as of 00:59, 27 September 2017
 Haynes Lab - Short Projects
|
 Main project page Previous entry Next entry
| |||||||||||||||||||||||||||||||||||||||||||||||||
Ben Nyer - 28/05/2015Cloning gRNAs into pSPgRNA for dCas9-HT binding to luc gene
Phosphorylate and anneal oligo pairs
Bio-Rad C1000 Touch Thermocycler: BbsI DigLig
| ||||||||||||||||||||||||||||||||||||||||||||||||||
