Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/04/15: Difference between revisions
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|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;">Transformation Results for 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}}}}]]}} | ||
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<!-- ##### 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== | ||
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Here are the colonies observed after one weekend grown at room temperature. I'll also be doing a plasmid miniprep of mCh_pSB1A3 so that I have more stocks of the cloning vector. | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Plate''' | |||
| align="center" style="background:#f0f0f0;"|'''# colonies''' | |||
|- | |||
| positive control||many dozens | |||
|- | |||
| sample||2 | |||
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| negative control||2 | |||
|} | |||
I'm going to set up two cultures for the two colonies on the sample plate, but am not very confident of the success of the ligation given the colonies on the negative control. | |||
==mCh_pSB1A3 Plasmid Miniprep== | |||
Decent yield for all four colonies picked. | |||
<b>--DNA Concentration--</b> | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Sample ID''' | |||
| align="center" style="background:#f0f0f0;"|'''Concentration (ng/µL)''' | |||
| align="center" style="background:#f0f0f0;"|'''St Dev (ng/µL)''' | |||
|- | |||
| mCh Vector 1||392.1||15.5 | |||
|- | |||
| mCh Vector 2||315.5||18.1 | |||
|- | |||
| mCh Vector 3||319.7||6.3 | |||
|- | |||
| mCh Vector 4||375.1||7.1 | |||
|} | |||
----- | |||
<b>--Restriction Digest--</b> | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Reagent''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume''' | |||
|- | |||
| DNA||600ng (1.9 - 1.5 µL) | |||
|- | |||
| 10x FastDigest Buffer||3 | |||
|- | |||
| EcoRI||1 | |||
|- | |||
| PstI||1 | |||
|- | |||
| Water||23 | |||
|- | |||
| Total||30 | |||
|} | |||
Digest for 15 minutes at 37°C. | |||
----- | |||
<b>--Gel Electrophoresis--</b> | |||
[[Image:2015-04-15_mCherry_vector_digest_annotated.png]] | |||
Expected size fragments from EcoRI/PstI digest: 2114, 980 | |||
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__NOTOC__ | __NOTOC__ |
Latest revision as of 00:54, 27 September 2017
Transformation Results for DBN001_pSB1A3 | Main project page Previous entry Next entry | |||||||||||||||||||||||||||||||||||||
OverviewHere are the colonies observed after one weekend grown at room temperature. I'll also be doing a plasmid miniprep of mCh_pSB1A3 so that I have more stocks of the cloning vector.
I'm going to set up two cultures for the two colonies on the sample plate, but am not very confident of the success of the ligation given the colonies on the negative control. mCh_pSB1A3 Plasmid MiniprepDecent yield for all four colonies picked. --DNA Concentration--
--Restriction Digest--
Digest for 15 minutes at 37°C.
--Gel Electrophoresis-- Expected size fragments from EcoRI/PstI digest: 2114, 980
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