Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2016/02/09: Difference between revisions

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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Today's project is...</span>
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Today's project is...</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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* ligation
* ligation
* transformation
* transformation
==Plasmid preps==
*HA1_v0120 1: 24 ng/µL
*HA1_v0120 2: 7 ng/µL
*DBN011 1: 124 ng/µL
*DBN011 2: 106 ng/µL
*DBN011 3: 27 ng/µL
*DBN011 4: 96 ng/µL
==Restriction Digests==
HA1 (E&S):<br>
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Reagent'''
| align="center" style="background:#f0f0f0;"|'''Volume (µL)'''
|-
| DNA||25
|-
| FD 10x GRN||3
|-
| EcoRI||1
|-
| Spe1||1
|-
|
|-
| Total||30
|}
DBN011 (E&X):<br>
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Reagent'''
| align="center" style="background:#f0f0f0;"|'''Volume (µL)'''
|-
| DNA||25
|-
| FD 10x GRN||3
|-
| EcoRI||1
|-
| XbaI||1
|-
|
|-
| Total||30
|}
Incubate at 37 °C for 10 minutes, then gel purify. HA1 part is 106 bp, DBN011 part is 5769 bp.
==Gel Extraction Results==
HA1 part looks good, but very faint band. DBN011 1, 2, and 4 look good. Proceeding to extract 1 and 2.
*HA1 (E&S digested): 4 ng/µL, 260/280 high (~3)
*DBN011 1 (E&X digested): 18.1 ng/µL
*DBN011 2 (E&X digested): 13.6 ng/µL
==Ligation==
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Reagent'''
| align="center" style="background:#f0f0f0;"|'''Volume (µL)'''
| align="center" style="background:#f0f0f0;"|'''Negative'''
|-
| Vector (DBN011 1) (50 ng)||2.8||2.8
|-
| Insert (HA1) (10:1)||2.3||0.0
|-
| 10x buffer||1.0||1.0
|-
| ligase||1.0||1.0
|-
| H2O||2.9||5.2
|-
| ||||
|-
| Total||10.0||10.0
|}
Incubate at room temperature for 10 minutes, then take 1 µL of reaction volume and use for quick transformation. Count colonies tomorrow.


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Latest revision as of 01:32, 27 September 2017

Today's project is... Main project page
Previous entry      Next entry

Overview

DBN011 ligation results:

  • Negative control: 8 colonies
  • Treatment: 18 colonies

Going to pick 4 colonies from the treatment plate and proceed with streaks & liquid cultures.

To do:

  • plasmid prep
  • DNA quantification
  • Restriction digest, HA1_v0120 with E&S (106 bp)
  • Restriction digest, DBN011 with E&X (5769 bp)
  • DNA quantification
  • ligation
  • transformation

Plasmid preps

  • HA1_v0120 1: 24 ng/µL
  • HA1_v0120 2: 7 ng/µL
  • DBN011 1: 124 ng/µL
  • DBN011 2: 106 ng/µL
  • DBN011 3: 27 ng/µL
  • DBN011 4: 96 ng/µL

Restriction Digests

HA1 (E&S):

Reagent Volume (µL)
DNA 25
FD 10x GRN 3
EcoRI 1
Spe1 1
Total 30

DBN011 (E&X):

Reagent Volume (µL)
DNA 25
FD 10x GRN 3
EcoRI 1
XbaI 1
Total 30

Incubate at 37 °C for 10 minutes, then gel purify. HA1 part is 106 bp, DBN011 part is 5769 bp.

Gel Extraction Results

HA1 part looks good, but very faint band. DBN011 1, 2, and 4 look good. Proceeding to extract 1 and 2.

  • HA1 (E&S digested): 4 ng/µL, 260/280 high (~3)
  • DBN011 1 (E&X digested): 18.1 ng/µL
  • DBN011 2 (E&X digested): 13.6 ng/µL

Ligation

Reagent Volume (µL) Negative
Vector (DBN011 1) (50 ng) 2.8 2.8
Insert (HA1) (10:1) 2.3 0.0
10x buffer 1.0 1.0
ligase 1.0 1.0
H2O 2.9 5.2
Total 10.0 10.0

Incubate at room temperature for 10 minutes, then take 1 µL of reaction volume and use for quick transformation. Count colonies tomorrow.



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