Haynes Lab:Notebook/Short Projects/2015/05/28: Difference between revisions

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|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"|<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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| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Rxn
| bgcolor=#cfcfcf | Rxn
| bgcolor=#cfcfcf | Mix (7x)
| bgcolor=#cfcfcf | Mix (5x)
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| 100 μM Oligo 1 || 1.0 || ---
| 100 μM Oligo 1 || 1.0 || ---
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| 100 μM Oligo 2 || 1.0 || ---
| 100 μM Oligo 2 || 1.0 || ---
|-
|-
| 10x T4 Lign buf (NEB) || 1.0 || 7.0
| 10x T4 Lign buf (NEB) || 1.0 || 5.0
|-
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| T4 PNK (NEB) || 0.5 || 3.5
| T4 PNK (NEB) || 0.5 || 2.5
|-
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| dH<sub>2</sub>O || 6.5 || 45.5
| dH<sub>2</sub>O || 6.5 || 32.5
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| &nbsp; || 10.0
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LabNet OptiMax Thermocycler: AnOlig RD
Bio-Rad C1000 Touch Thermocycler: Olig PNK-Ann
* 37°C, 30 min
* 37°C, 30 min
* 95°C, 5 min
* 95°C, 5 min
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| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Rxn
| bgcolor=#cfcfcf | Rxn
| bgcolor=#cfcfcf | Mix (x7)
| bgcolor=#cfcfcf | Mix (x5)
|-
|-
| 100 ng Vector || 0.5 || 3.5
| 100 ng Vector || 0.5 || 2.5
|-
|-
| 1:250 dsOligo || 2.0 || ---
| 1:250 dsOligo || 2.0 || ---
|-
|-
| 10x FD buf || 2.0 || 14.0
| 10x FD buf || 2.0 || 10.0
|-
|-
| 10 mM DTT || 1.0 || 7.0
| 10 mM DTT || 1.0 || 5.0
|-
|-
| 10 mM ATP || 1.0 || 7.0
| 10 mM ATP || 1.0 || 5.0
|-
|-
| FastDigest BbsI || 1.0 || 7.0
| FastDigest BbsI || 1.0 || 5.0
|-
|-
| T4 DNA ligase || 0.5 || 3.5
| T4 DNA ligase || 0.5 || 2.5
|-
|-
| dH<sub>2</sub>O || 12.0 || 84.0
| dH<sub>2</sub>O || 12.0 || 60.0
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| &nbsp; || 20.0
| &nbsp; || 20.0
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LabNet OptiMax Thermocycler: BbsI Dig/Lig
Bio-Rad C1000 Touch Thermocycler: BbsI DigLig
* 6x [37°C, 5 min; 23°C, 5 min]
* 6x [37°C, 5 min; 23°C, 5 min]
* 4°C, ∞
* 4°C, ∞

Latest revision as of 00:59, 27 September 2017

Haynes Lab - Short Projects Main project page
Previous entry      Next entry

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

  1. g025 T/B
  2. g031 T/B
  3. g034 T/B
  4. g048 T/B
Reagent Rxn 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
dH2O 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 dH2O


Digestion/ Ligation Reactions (Vector / dsOligo Insert)

  1. pSPgRNA / g025
  2. pSPgRNA / g031
  3. pSPgRNA / g034
  4. pSPgRNA / g048
Reagent Rxn 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
dH2O 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


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