Haynes Lab:Notebook/Engineering PC-TFs/2014/04/18: Difference between revisions

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|style="background-color: #800000" 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>}}
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==Summary==
==Summary==
*  
4/16
*Cloned mv9cmv construct into DH5a-T cells using standard procedure. Incubated overnight.


4/17
* Removed plates from incubator.
{| {{table}} border="1" cellspacing="3" <!-- Plates. table -->
|- valign="top"
| rowspan="7" | [[Image:2014-04-17 15.36.21.jpg|400px|Hover name]];
|- valign="top"
| rowspan="7" | [[Image:2014-04-17 15.36.39.jpg|400px|Hover name]]<br>;
|- valign="top"
|}
*Plate 1 is the experimental plate with growth. Plate 2 is the negative control and had no growth (debris is visible underneath the plate).


4/18
*Grew up liquid cultures from prior night.
*Miniprepped cultures.
{| {{table}}
|- bgcolor=#cfcfcf
| Plasmid || OD260 || OD260/280 || ng/μL
|-
| 1. Colony 1  || 0.202 || 1.905 || 162
|-
| 2. Colony 2  || 0.253 || 1.92 || 203
|}
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|}
|}


*Restriction Digest
{| {{table}} border="1" cellspacing="3"
<!-- Editing: the coding for this table is a bit more advanced. -->
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<!-- The | symbols on the next two lines start new cells in the same row. This does the same thing as ||, but you have to use | on new lines to set formatting for cells. -->
<!-- bgcolor=#cfcfcf *colors* the *background* of the Reagent and Volume cells grey.  -->
<!-- The next two "rowspan=7" cells span  all 7 rows in the table so that they can fit the "Expected" list and a gel image. rowspan is how you create merged rows in Wiki code. -->
<!-- Replace Plasmid 1 and 2 with the names of your plasmids. Replace insert size and vector size with appropriate information for your plasmids. If you only have one Plasmid, delete all the text for Plasmid 2
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|- valign="top"
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume
| rowspan="7" | <u>Expected:</u><br>1. CMV = 643bp <br>
|-
| DNA(CMV/MV9) || 4.0 μL
|-
| 10X buffer || 1.5 μL
|-
| NotI || 1.0 μL
|-
| NruI || 1.0 μL
|-
| dH<sub>2</sub>O || 7.5 μL
|-
| &nbsp; || 15 μL --> 37°C/ 15 min.
|}
*Verified for the sake of not wasting money on sequencing in the case that something unexpected occurred.
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
|- valign="top"
| rowspan="7" | [[Image:2014-04-18 13.13.10.jpg|400px|Hover name]]<br>15 μL/lane; 1% agarose;
[http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
|}
*Expected base pair length: 643
*Observed base pair length: ~650
<br> <br>
*Sent off for sequencing
{| {{table}}
|- bgcolor=#cfcfcf
| Plasmid Title || Primer || Water || Plasmid (~200ng/uL)
|-
| 1. cmvmv9f1  || FP 1uL DD122 B || 8uL || 1uL
|-
| 2. cmvmv9r1  || RP 1uL DD123 B || 8uL || 1uL
|}
__NOTOC__
__NOTOC__

Latest revision as of 23:54, 26 September 2017



 		Main project page
Previous entry      Next entry

Summary

4/16

  • Cloned mv9cmv construct into DH5a-T cells using standard procedure. Incubated overnight.

4/17

  • Removed plates from incubator.
Hover name;
Hover name
;
  • Plate 1 is the experimental plate with growth. Plate 2 is the negative control and had no growth (debris is visible underneath the plate).

4/18

  • Grew up liquid cultures from prior night.
  • Miniprepped cultures.
Plasmid OD260 OD260/280 ng/μL
1. Colony 1 0.202 1.905 162
2. Colony 2 0.253 1.92 203
  • Restriction Digest
Reagent Volume Expected:
1. CMV = 643bp
DNA(CMV/MV9) 4.0 μL
10X buffer 1.5 μL
NotI 1.0 μL
NruI 1.0 μL
dH2O 7.5 μL
  15 μL --> 37°C/ 15 min.
  • Verified for the sake of not wasting money on sequencing in the case that something unexpected occurred.
Hover name
15 μL/lane; 1% agarose; 
Ladder
  • Expected base pair length: 643
  • Observed base pair length: ~650



  • Sent off for sequencing
Plasmid Title Primer Water Plasmid (~200ng/uL)
1. cmvmv9f1 FP 1uL DD122 B 8uL 1uL
2. cmvmv9r1 RP 1uL DD123 B 8uL 1uL
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