Haynes Lab:Notebook/Short Projects/2015/02/23: Difference between revisions

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==02/23/15 - Alexander Ellingson==
==02/23/15 - Alexander Ellingson==


<!-- Replace #name# with activation domain name.  
<!-- Replace #name# with activation domain name. -->
<!-- Replace #size# with the length of this activation domain in base pairs. -->
<!-- Replace #f-primer# with name of the forward primer -->
<!-- Replace #r-primer# with name of the reverse primer -->


'''PCR-verify fragment primers'''<br>
'''PCR-verify activation domain primers'''<br>
Try different cDNA libraries for the #name# fragment to account for any mutations or deletions
Try different cDNA libraries for the #name# fragment to account for any mutations or deletions
# U2OS C002, 1:1000 cDNA dilution; #name# = #size#: ATF2_f1/ ATF2_r1
# U2OS C002, 1:1000 cDNA dilution; #name# = #size#: #f-primer#/ #r-primer#
# SKNSH C001, 1:1000 cDNA dilution; same
# SKNSH C001, 1:1000 cDNA dilution; same
# K562 C001, 1:1000 cDNA dilution; same
# K562 C001, 1:1000 cDNA dilution; same
Line 23: Line 26:
| bgcolor=#cfcfcf | Rxn2
| bgcolor=#cfcfcf | Rxn2
| bgcolor=#cfcfcf | Rxn3
| bgcolor=#cfcfcf | Rxn3
| bgcolor=#cfcfcf | Rxn4
| rowspan="7" | Expected:<br>1. U2OS #name# = #size#<br>2. SKNSH #name# = #size#<br>2. K562 #name# = #size#
| rowspan="7" | Expected:<br>1. KAH60 = 6637<br>2. ATF2 = 906<br>3. ATF2 = 906<br>4. ATF2 = 906
| rowspan="7" | [[Image:SomeGel.jpg|270px|Hover name]]<br>10 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
| rowspan="7" | [[Image:KAH012315_gel.jpg|270px|Hover name]]<br>10 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
|-
|-
| Template || 0.2 || 1.0 || 1.0 || 1.0
| Template || 1.0 || 1.0 || 1.0
|-
|-
| 10 uM fwd primer || 1.0 || 1.0 || 1.0 || 1.0
| 10 uM fwd primer || 1.0 || 1.0 || 1.0  
|-
|-
| 10 uM rev primer || 1.0 || 1.0 || 1.0 || 1.0
| 10 uM rev primer || 1.0 || 1.0 || 1.0 || 1.0
Line 35: Line 37:
| 2x GoTaq green || 12.5 || 12.5 || 12.5 || 12.5  
| 2x GoTaq green || 12.5 || 12.5 || 12.5 || 12.5  
|-
|-
| dH<sub>2</sub>O || 10.3 || 9.5 || 9.5 || 9.5
| dH<sub>2</sub>O || 9.5 || 9.5 || 9.5
|-
|-
| &nbsp; || 25.0 || 25.0 || 25.0 || 25.0
| &nbsp; || 25.0 || 25.0 || 25.0
|}
|}


Program: GOTAQ35cyc
Program: GOTAQ35cyc
* 95°C, 3 min
* 95°C, 3 min
* 30x[95°C, 1 min; 57°C, 1 min; 72°C, 3 min]
* 35x[95°C, 1 min; 57°C, 1 min; 72°C, 3 min]
* 72°C, 3 min
* 72°C, 3 min
* 4°C ∞
* 4°C ∞

Revision as of 11:23, 23 February 2015

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02/23/15 - Alexander Ellingson

PCR-verify activation domain primers
Try different cDNA libraries for the #name# fragment to account for any mutations or deletions

  1. U2OS C002, 1:1000 cDNA dilution; #name# = #size#: #f-primer#/ #r-primer#
  2. SKNSH C001, 1:1000 cDNA dilution; same
  3. K562 C001, 1:1000 cDNA dilution; same


Reagent Rxn1 Rxn2 Rxn3 Expected:
1. U2OS #name# = #size#
2. SKNSH #name# = #size#
2. K562 #name# = #size# 		Hover name
10 μL/lane, 1% agarose; Ladder
Template 1.0 1.0 1.0
10 uM fwd primer 1.0 1.0 1.0
10 uM rev primer 1.0 1.0 1.0 1.0
2x GoTaq green 12.5 12.5 12.5 12.5
dH2O 9.5 9.5 9.5
  25.0 25.0 25.0

Program: GOTAQ35cyc

  • 95°C, 3 min
  • 35x[95°C, 1 min; 57°C, 1 min; 72°C, 3 min]
  • 72°C, 3 min
  • 4°C ∞




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