Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/04/16: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
(fix raw html notebook nav) |
|||
| (6 intermediate revisions by one other user not shown) | |||
| Line 1: | Line 1: | ||
{|{{table}} width="800" | {|{{table}} width="800" | ||
|- | |- | ||
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Plasmid Miniprep of DBN001_pSB1A3</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. ##### --> | ||
== | ==Overview== | ||
<!-- Precede finished items with a checkmark ✓ --> | <!-- Precede finished items with a checkmark ✓ --> | ||
I'll be taking the two colonies from the sample plate and growing them up for plasmid miniprep. | |||
==Notes== | |||
Both colonies grown in liquid culture are normal colored (not red). Cell density appears low though, with lots of sticky filaments. Indication that they were not subjected to continuous shaking overnight. | Both colonies grown in liquid culture are normal colored (not red). Cell density appears low though, with lots of sticky filaments. Indication that they were not subjected to continuous shaking overnight. | ||
| Line 16: | Line 18: | ||
==DNA Concentration== | |||
{| {{table}} | {| {{table}} | ||
| Line 24: | Line 25: | ||
| align="center" style="background:#f0f0f0;"|'''St Dev (ng/µL)''' | | align="center" style="background:#f0f0f0;"|'''St Dev (ng/µL)''' | ||
|- | |- | ||
| | | DBN001_pSB1A3, colony 1||56.7||2.6 | ||
|- | |- | ||
| | | DBN001_pSB1A3, colony 2||54.0||0.2 | ||
|} | |} | ||
==Restriction Digest== | |||
{| {{table}} | {| {{table}} | ||
| Line 51: | Line 51: | ||
Digest for 15 minutes at 37°C. | Digest for 15 minutes at 37°C. | ||
<b>NOTE:</b> Should've used SpeI, not PstI. Oh well, won't make much of a difference for analysis of the sequence. | |||
==Gel Electrophoresis== | |||
Expected size fragments from EcoRI/PstI digestion: 2114, 189, 53 | |||
[[Image:2015-04-16_DBN001_pSB1A3_digest_annotated.png]] | |||
No trace of plasmid, just a smear of (presumably) genomic DNA. Will have to repeat plasmid prep and hope for a higher yield. | |||
Latest revision as of 00:54, 27 September 2017
 Plasmid Miniprep of DBN001_pSB1A3
|
 Main project page Previous entry Next entry
| |||||||||||||||||||||||
OverviewI'll be taking the two colonies from the sample plate and growing them up for plasmid miniprep. NotesBoth colonies grown in liquid culture are normal colored (not red). Cell density appears low though, with lots of sticky filaments. Indication that they were not subjected to continuous shaking overnight. Also, for future reference: when performing digestion/ligation, set up a control plate using linearized vector treated with phosphatase. Presence of colonies on this plate indicates that the phosphatase step did not completely work.
DNA Concentration
Restriction Digest
Digest for 15 minutes at 37°C. NOTE: Should've used SpeI, not PstI. Oh well, won't make much of a difference for analysis of the sequence.
Gel ElectrophoresisExpected size fragments from EcoRI/PstI digestion: 2114, 189, 53
No trace of plasmid, just a smear of (presumably) genomic DNA. Will have to repeat plasmid prep and hope for a higher yield.
| ||||||||||||||||||||||||
