Difference between revisions of "Haynes Lab:Notebook/Engineering PC-TFs/2012/12/07"

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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Engineering PC-TFs</span>
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|style="background-color: #FFCC00"|<br>[[Image:Hayneslab3.gif|200px|center]][[Image:Asu logo 3.gif|200px|center]]<span style="font-size:22px;"><br></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>}}
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|style="background-color: #800000" 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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==Summary==
 
==Summary==
*  
+
* Performed another PCR run because lid was not closed entirely yesterday. Next time in lab(Monday), confirmation gel will be run to show that PCR rxns worked.
  
  
 +
*Assemblies
 +
 +
1. hPCD - Pflex - BL01<br>
 +
2. hPCD - BL01
 +
 +
{|border="1" cellpadding="5" cellspacing="0"
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|-
 +
! scope="col" style="background:#efefef;" | #
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! scope="col" style="background:#efefef;" | Template
 +
! scope="col" style="background:#efefef;" | Primers
 +
|-
 +
|1
 +
|hPCD
 +
|9, 2
 +
|-
 +
|2
 +
|Plflex
 +
|3, 4
 +
|-
 +
|3
 +
|BL01
 +
|5,10
 +
|-
 +
|4
 +
|hPCD
 +
|9, 7
 +
|-
 +
|5
 +
|BL01
 +
|8,10
 +
|-
 +
|}
 +
 +
{|border="1" cellpadding="5" cellspacing="0"
 +
|-
 +
! scope="col" style="background:#efefef;" |
 +
! scope="col" style="background:#efefef;" | μL
 +
|-
 +
|Template
 +
|.5 μL
 +
|-
 +
|Primer 1 (10 μm)
 +
|1.0 μL
 +
|-
 +
|Primer 2 (10 μm)
 +
|1.0 μL
 +
|-
 +
|2*GoTaq
 +
|12. 5
 +
|-
 +
|DH2O
 +
|10.0 μL
 +
|-
 +
|}
 +
PCR was set with preset settings:
 +
 +
[[Image:pcrsett.jpg|300px]]
 +
 +
----
 +
*Ran Zymoclean DNA recovery kit.
 +
 +
*Add 3 volumes of ADB Buffer to each volume of gel. (Each gel slice is assumed to be 200 mg)
 +
*Therefore, add 600 μL of ADB Buffer.
 +
*Incubate at 55°C for 10 minutes.
 +
*Load melted agarose solution into Spin Column in a collection tube.
 +
*Centrifuge at max speed for 30 seconds.
 +
*Discard flow-through.
 +
*Add 200 μL of DNA wash buffer to the spin columns and centrifuge for 30 seconds.
 +
*Place spin column in a 1.5 mL tube. Add 10μL of DNA Elution Buffer to elute the DNA.
 +
*Place tube into freezer box.
 +
 +
''There was not much DNA that came from the purification kit. A reason for this is because the bands were smaller than usual, according to Vi. Vi is running another gel digest to cut the backbone from the PSB1A3 vector. Rene said she was also running into problems with minipreping the vectors and cutting it. A follow-up with Dr. Haynes will confirm next step and possible options.
 
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|}
 
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__NOTOC__
 
__NOTOC__

Latest revision as of 21:19, 26 September 2017


Hayneslab3.gif
Asu logo 3.gif

Report.pngMain project page
Resultset previous.pngPrevious entry      Next entryResultset next.png

Summary

  • Performed another PCR run because lid was not closed entirely yesterday. Next time in lab(Monday), confirmation gel will be run to show that PCR rxns worked.


  • Assemblies

1. hPCD - Pflex - BL01
2. hPCD - BL01

# Template Primers
1 hPCD 9, 2
2 Plflex 3, 4
3 BL01 5,10
4 hPCD 9, 7
5 BL01 8,10
μL
Template .5 μL
Primer 1 (10 μm) 1.0 μL
Primer 2 (10 μm) 1.0 μL
2*GoTaq 12. 5
DH2O 10.0 μL

PCR was set with preset settings:

Pcrsett.jpg


  • Ran Zymoclean DNA recovery kit.
  • Add 3 volumes of ADB Buffer to each volume of gel. (Each gel slice is assumed to be 200 mg)
  • Therefore, add 600 μL of ADB Buffer.
  • Incubate at 55°C for 10 minutes.
  • Load melted agarose solution into Spin Column in a collection tube.
  • Centrifuge at max speed for 30 seconds.
  • Discard flow-through.
  • Add 200 μL of DNA wash buffer to the spin columns and centrifuge for 30 seconds.
  • Place spin column in a 1.5 mL tube. Add 10μL of DNA Elution Buffer to elute the DNA.
  • Place tube into freezer box.

There was not much DNA that came from the purification kit. A reason for this is because the bands were smaller than usual, according to Vi. Vi is running another gel digest to cut the backbone from the PSB1A3 vector. Rene said she was also running into problems with minipreping the vectors and cutting it. A follow-up with Dr. Haynes will confirm next step and possible options.