Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2016/02/09: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
(fix raw html notebook nav) |
|||
(4 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;">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"| | |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 19: | Line 19: | ||
* DNA quantification | * DNA quantification | ||
* Restriction digest, HA1_v0120 with E&S (106 bp) | * Restriction digest, HA1_v0120 with E&S (106 bp) | ||
* Restriction digest, DBN011 with E&X ( | * Restriction digest, DBN011 with E&X (5769 bp) | ||
* DNA quantification | * DNA quantification | ||
* 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. | |||
<!-- ##### 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 01:32, 27 September 2017
Today's project is... | Main project page Previous entry Next entry | ||||||||||||||||||||||||||||||||||||||||||||||||||
OverviewDBN011 ligation results:
Going to pick 4 colonies from the treatment plate and proceed with streaks & liquid cultures. To do:
Plasmid preps
Restriction DigestsHA1 (E&S):
DBN011 (E&X):
Incubate at 37 °C for 10 minutes, then gel purify. HA1 part is 106 bp, DBN011 part is 5769 bp. Gel Extraction ResultsHA1 part looks good, but very faint band. DBN011 1, 2, and 4 look good. Proceeding to extract 1 and 2.
Ligation
Incubate at room temperature for 10 minutes, then take 1 µL of reaction volume and use for quick transformation. Count colonies tomorrow.
|