Haynes Lab:Notebook/Short Projects/2015/03/01: Difference between revisions

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(Autocreate 2015/03/01 Entry for Haynes_Lab:Notebook/Short_Projects)
 
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==02/23/15 - Alexander Ellingson==
==Karmella Haynes - 03/01/15==


'''PCR-verify activation domain primers'''<br>
* Passaging - KAH126, split 1:10
Try different cDNA libraries for the CARM1, P300, and MYB fragment to account for any mutations or deletions
* 6-well plate - KAH126
# U2OS C002, 1:1000 cDNA dilution; CARM1 = 1827 bp: CARM1 F1/ CARM1 R1; P300 = 1848 bp: P300 F1/ P300 R1; MYB = 159 bp: MYB F1/ MYB R1.
* Dox induction - KAH126
# SKNSH C001, 1:1000 cDNA dilution; same.
# K562 C001, 1:1000 cDNA dilution; same.


----


{| {{table}} cellspacing="3" <!-- PCR rxn. table -->
'''Passaging - KAH126'''
|- valign="top"
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Rxn1
| bgcolor=#cfcfcf | Rxn2
| bgcolor=#cfcfcf | Rxn3
| rowspan="7" | Expected:<br>1. U2OS CARM1 = 1827 bp<br>1. U2OS P300 = 1848 bp<br>1. U2OS MYB = 159 bp<br>2. SKNSH CARM1 = 1827 bp<br>2. SKNSH P300 = 1848 bp<br>2. SKNSH MYB = 159 bp<br>3. K562 CARM1 = 1827 bp<br>3. K562 P300 = 1848 bp<br>3. K562 MYB = 159 bp
| 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]
|-
| 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
|-
| 2x GoTaq green || 12.5 || 12.5 || 12.5
|-
| dH<sub>2</sub>O || 9.5 || 9.5 || 9.5
|-
| &nbsp; || 25.0 || 25.0 || 25.0
|}


Program: GOTAQ35cyc
* Protocol:  
* 95°C, 3 min
* Use 90-100% confluent '''T-75'''
* 35x[95°C, 1 min; 57°C, 1 min; 72°C, 3 min]
 
* 72°C, 3 min
----
* 4°C ∞
'''6-well plate - KAH126'''
 
* Start with 90-100% confluent '''T-75''' = 10E6/10 mL = 1.0E6/1 mL
*  




Revision as of 15:17, 25 March 2015

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Karmella Haynes - 03/01/15

  • Passaging - KAH126, split 1:10
  • 6-well plate - KAH126
  • Dox induction - KAH126

Passaging - KAH126

  • Protocol:
  • Use 90-100% confluent T-75

6-well plate - KAH126

  • Start with 90-100% confluent T-75 = 10E6/10 mL = 1.0E6/1 mL



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