Haynes Lab:Notebook/Engineering PC-TFs/2012/12/06: Difference between revisions

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{|{{table}} width="800"
{|{{table}} width="800"
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Engineering PC-TFs</span>
|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>}}
|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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Dilute primers that came from IDT (10x dilution of concentration)
Dilute primers that came from IDT (10x dilution of concentration)


BL_Primer09 26.6 nm --> 266 μL dilution
*BL_Primer09 26.6 nm --> 266 μL dilution
BL_Primer10 25.7 nm --> 257 μL dilution
*BL_Primer10 25.7 nm --> 257 μL dilution


*Thaw and dilute primers that came  (x10 dilution)
*Thaw and dilute primers that came  (x10 dilution)
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*10 μL primer stock
*10 μL primer stock


(10 μm in each tube for PCR reaction)
(10 μm in each tube for PCR reaction)<br>
 
______________________________________
*Assemblies
*Assemblies


1. hPCD - Pflex - BL01<br>
1. hPCD - Pflex - BL01<br>
2. hPCD - BL01
2. hPCD - BL01
{|border="1" cellpadding="5" cellspacing="0"
|-
! scope="col" style="background:#efefef;" | #
! 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
|-
|}
Run PCR with preset GoTaq settings. Need to ask Dr. Haynes to be certain settings are correct for my reactions.<br>
______________________
*Digest the pSB1A2/3 plasmid (from Rene) with XbaI & SpeI to get rid of the insert
{| class="wikitable" border="0" cellspacing="3" <!-- Digest rxn. table -->
|-valign="top"
| <u>Reagent</u> || <u>Volume</u> || &nbsp;
| rowspan="9" |
|-
| DNA (plasmid) || 20.0
|-
| 10x buffer || 3.0
|-
| XbaI || 1.0
|-
| SpeI || 1.0
|-
| dH<sub>2</sub>O || 5.0
|-
| &nbsp; || 30 μL --> 37°C/ ~10 min.
|}
*Vi helped me with running gel because I had class. Vi will put digest into gel and will get results.
*''I believe that I did not wait long enough after I microwaved the gel to put in the ethnium bromide. I need to be sure that I wait until its cooled down.''
<br style="clear:both;"/>
----
[[Image:December_6_2012.jpg|400px|thumb|PSB1A3 VECTOR digest]]
*Vi cut gel and has it available for me to work on tomorrow afternoon.
*Vi also took out my PCR reaction. She told me that the lid was not closed all the way, so I may need to redo rxns.




Revision as of 18:32, 6 September 2013



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Summary

Dilute primers that came from IDT (10x dilution of concentration)

  • BL_Primer09 26.6 nm --> 266 μL dilution
  • BL_Primer10 25.7 nm --> 257 μL dilution
  • Thaw and dilute primers that came (x10 dilution)
  • 90 μL water
  • 10 μL primer stock

(10 μm in each tube for PCR reaction)
______________________________________

  • 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

Run PCR with preset GoTaq settings. Need to ask Dr. Haynes to be certain settings are correct for my reactions.
______________________ 

*Digest the pSB1A2/3 plasmid (from Rene) with XbaI & SpeI to get rid of the insert
Reagent Volume  
DNA (plasmid) 20.0
10x buffer 3.0
XbaI 1.0
SpeI 1.0
dH2O 5.0
  30 μL --> 37°C/ ~10 min.
  • Vi helped me with running gel because I had class. Vi will put digest into gel and will get results.
  • I believe that I did not wait long enough after I microwaved the gel to put in the ethnium bromide. I need to be sure that I wait until its cooled down.


PSB1A3 VECTOR digest
  • Vi cut gel and has it available for me to work on tomorrow afternoon.
  • Vi also took out my PCR reaction. She told me that the lid was not closed all the way, so I may need to redo rxns.



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