Haynes Lab:Notebook:Shared/Cloning Bootcamp/2015/11/20: Difference between revisions

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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Haynes Lab Cloning Bootcamp</span>
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Haynes Lab Cloning Bootcamp</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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<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
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==Baylee Murphy- 11/20/15==
==Baylee Murphy- 11/20/15 Session 3==


KAH126- MV2 Culture inoculation results from [http://openwetware.org/wiki/Haynes_Lab:Notebook:Shared/Cloning_Bootcamp/2015/11/18 Session 2]
KAH126- MV2 Culture inoculation results from [http://openwetware.org/wiki/Haynes_Lab:Notebook:Shared/Cloning_Bootcamp/2015/11/18 Session 2]
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*(2) Sample 2- KAH126-MV2 colony 2, [BB#}; part = [length in bp]; vector= [length in bp]
*(2) Sample 2- KAH126-MV2 colony 2, [BB#}; part = [length in bp]; vector= [length in bp]


Results from NanoDrop
====DNA concentration Data====
 
{|
| align="center" style="background:#f0f0f0;"|'''Sample Read#'''
| align="center" style="background:#f0f0f0;"|'''Location'''
| align="center" style="background:#f0f0f0;"|'''260 Raw'''
| align="center" style="background:#f0f0f0;"|'''280 Raw'''
| align="center" style="background:#f0f0f0;"|'''320 Raw'''
| align="center" style="background:#f0f0f0;"|'''260'''
| align="center" style="background:#f0f0f0;"|'''280'''
| align="center" style="background:#f0f0f0;"|'''260/280'''
| align="center" style="background:#f0f0f0;"|'''ng/µL'''
|-
| BM KAH126-1||C2||0.32||0.207||0.08||0.249||0.131||1.909||249.344
|-
| BM KAH126-1||C3||0.313||0.201||0.074||0.245||0.128||1.911||244.882
|-
| BM KAH126-2||D2||0.323||0.203||0.06||0.272||0.146||1.861||272.416
|-
| BM KAH126-2||D3||0.319||0.2||0.066||0.258||0.136||1.904||258.244
|}


'''Excel table'''




Using Xba restriction enzyme which will preform 2 cuts- expending bands at 1934 bp and 4505 bp


'''insert picture'''
=Plasmid Validation=




'''Restriction Digest Table'''<br>
* Checked plasmid minipreps with Xba digest


'''plasmid validation template page'''
{| {{table}} border="1" cellspacing="3"
<!-- Editing: the coding for this table is a bit more advanced. -->
<!-- valign="top" aligns all the text in the first row to the top.  -->
<!-- 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
<!-- After you upload your gel image to OWW, replace "GelImage.jpg" with the name of your image file -->
<!-- &nbsp; is ASCII code for an invisible space. -->
|- valign="top"
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume
| rowspan="7" | <u>Expected:</u><br>1. KAH126-MV2 = 1934 bp and 4505 bp<br>
| rowspan="7" | [[Image:Screen Shot 2015-11-21 at 4.30.33 PM.png|400px|Today's gel]]<br>10 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
|-
| DNA(plasmid) || 1.0 μL
|-
| 10X buffer || 1.5 μL
|-
| Xba || 1.0 μL
|-
| dH<sub>2</sub>O || 10.5 μL
|-
| &nbsp; || 10 μL --> 37°C/ 20 min.
|}
 
====Control for 1 and 2====
 
 
{| {{table}} border="1" cellspacing="3"
<!-- Editing: the coding for this table is a bit more advanced. -->
<!-- valign="top" aligns all the text in the first row to the top.  -->
<!-- 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
<!-- After you upload your gel image to OWW, replace "GelImage.jpg" with the name of your image file -->
<!-- &nbsp; is ASCII code for an invisible space. -->
|- valign="top"
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume
|-
| DNA(plasmid) || 1.0 μL
|-
| 10X buffer || 1.5 μL
|-
| dH<sub>2</sub>O || 11.5 μL
|}
 
 
 
Results of restriction digest show expected bands and indicate Xba enzyme worked


KAH126 incluswa Hpdc, scar, mcherry: about 1000 bp
KAH126 incluswa Hpdc, scar, mcherry: about 1000 bp
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'''DNA Sequencing Samples'''
<!-- * signs create an automatically bulleted list. Replace 'date' with the date you submitted the samples -->
<!-- The Order Number is shown at the top of your sequencing order form. Record in the indicated spot below -->
* Submitted to DNASU on: '11/20/15'
* Order Number:
<!-- # signs create an automatically numbered list. Replace Plasmid 1 & 2 with the names of your plasmids. Replace 'name' with the appropriate name of each primer. -->
#Sample 1- KAH126-MV2 colony 1, forward primer DD122
#Sample 2- KAH126-MV2 colony 1, reverse primer DD123
#Sample 3- KAH126-MV2 colony 2, forward primer DD122
#Sample 4- KAH126-MV2 colony 2, reverse primer DD123


====Samples sent for sequencing at Bio Design around 4:45pm ====
*(1) Sample 1- KAH126-MV2 colony 1, forward primer DD122
*(2) Sample 2- KAH126-MV2 colony 1, reverse primer DD123
*(3) Sample 3- KAH126-MV2 colony 2, forward primer DD122
*(4) Sample 4- KAH126-MV2 colony 2, reverse primer DD123


<!--TEMPLATE CODE-->




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<!-- If you need to create an entry and someone else has already used this page, add another ==Name - Date== header below their entry and click save. -->


|}


__NOTOC__
__NOTOC__

Latest revision as of 01:20, 27 September 2017

Haynes Lab Cloning Bootcamp Main project page
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Baylee Murphy- 11/20/15 Session 3

KAH126- MV2 Culture inoculation results from Session 2

  • Liquid cultures 1 and 2 both had cloudy yellow broth, indicating growth
  • Streaks for both showed colony growth

Samples for mini prep and analysis

  • (1) Sample 1- KAH126-MV2 colony 1, [BB#}; part = [length in bp]; vector= [length in bp]
  • (2) Sample 2- KAH126-MV2 colony 2, [BB#}; part = [length in bp]; vector= [length in bp]

DNA concentration Data

Sample Read# Location 260 Raw 280 Raw 320 Raw 260 280 260/280 ng/µL
BM KAH126-1 C2 0.32 0.207 0.08 0.249 0.131 1.909 249.344
BM KAH126-1 C3 0.313 0.201 0.074 0.245 0.128 1.911 244.882
BM KAH126-2 D2 0.323 0.203 0.06 0.272 0.146 1.861 272.416
BM KAH126-2 D3 0.319 0.2 0.066 0.258 0.136 1.904 258.244



Plasmid Validation

Restriction Digest Table

  • Checked plasmid minipreps with Xba digest
Reagent Volume Expected:
1. KAH126-MV2 = 1934 bp and 4505 bp
 Today's gel
10 μL/lane; 1% agarose; Ladder
DNA(plasmid) 1.0 μL
10X buffer 1.5 μL
Xba 1.0 μL
dH2O 10.5 μL
  10 μL --> 37°C/ 20 min.

Control for 1 and 2

Reagent Volume
DNA(plasmid) 1.0 μL
10X buffer 1.5 μL
dH2O 11.5 μL


Results of restriction digest show expected bands and indicate Xba enzyme worked

KAH126 incluswa Hpdc, scar, mcherry: about 1000 bp

  • forward primer- DD122 10 uM
  • reverse primer-DD123 10 uM


DNA Sequencing Samples

  • Submitted to DNASU on: '11/20/15'
  • Order Number:
  1. Sample 1- KAH126-MV2 colony 1, forward primer DD122
  2. Sample 2- KAH126-MV2 colony 1, reverse primer DD123
  3. Sample 3- KAH126-MV2 colony 2, forward primer DD122
  4. Sample 4- KAH126-MV2 colony 2, reverse primer DD123
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