Haynes Lab:Notebook/Investigating Photo-Switchable Synthetic Nucleosomes/2012/11/06: Difference between revisions

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{|border="1" cellpadding="5" cellspacing="0" align="center"
{|border="1" cellpadding="5" cellspacing="0" align="center"
|+Restriction Digest Reagents
  |-
  |-
  ! scope="col" style="background:#efefef;" | Reagent
  ! scope="col" style="background:#efefef;" | Reagent
  ! scope="col" style="background:#efefef;" | H2B-GFP/EcoRI
  ! scope="col" style="background:#efefef;" | H2B-GFP/EcoRI
  ! scope="col" style="background:#efefef;" | H2B-GFP/PstI
  ! scope="col" style="background:#efefef;" | H2B-GFP/PstI
  ! scope="col" style="background:#efefef;" | LOV/EcoRI
  ! scope="col" style="background:#efefef;" | LOV/PstI
  |-
  |-
  |DNA(plasmid)
  |DNA(plasmid)
  |2.0  
  |2.0
  |2.0
  |2.0
  |2.0
  |2.0
  |-
  |-
Line 27: Line 28:
  |-
  |-
  |EcoRI
  |EcoRI
  |1
  |1.0
  |---
  |0
  |1
  |0
  |-
  |-
  |PstI
  |PstI
  |---
  |0
  |1
  |1.0
  |---
|1.0
  |-
  |dH<sub>2</sub>O
  |dH<sub>2</sub>O
  |10.5
  |10.5
Line 41: Line 43:
  |-
  |-
  |Total
  |Total
  |15
  |15.0
  |15
  |15.0
  |15
  |15.0
  |}
  |}


[[Image:Vi110212_gel1.tif|150px|Digest check 11/02/12]
 
{| class="wikitable" border=0
|-
| <u>Expected:</u><br>1. H2B-GFP/ EcoRI = 4885, 210 <br>2. H2B-GFP/ PstI = 5101<br>3. LOV/PstI = 251
| <br>
|}
 
<center>
[[Image:VN_Gel_11-6-12.png|150px|Results of the digests are shown.]]
[[Image:KAH_Fermentas_GeneRuler_1kbplus.jpg]]
</center>
 
<center>
Results of the digests are shown.
</center>
 
----
*'''[[User:Karmella Haynes|---Karmella]] 16:01, 7 November 2012 (EST)''': Note: Please re-do LOV/PstI
<br>
 
*'''[[User:Karmella Haynes|---Karmella]] 16:01, 7 November 2012 (EST)''': Here is a reaction table for PCR
 
* First, resuspend dry primer oligos (in blue-capped tubes) to 100 μM
* Then make working solutions of 10 μM
* Reactions:
 
# H2B-GFP plasmid + BB_H2B fwd + BB_H2B rev
# LOV plasmid + BB_LOV fwd + BB_LOV rev
 
{| class="wikitable" border=1 cellpadding="5" cellspacing="0"
|-
| Reagents || H2B || LOV
|-
| Plasmid DNA || 0.5 μL || 0.5
|-
| primer 1 (10 μM) || 1.0 || 1.0
|-
| primer 2 (10 μM) || 1.0 || 1.0
|-
| 2x GoTaq mix || 12.5 || 12.5
|-
| dH<sub>2</sub>O || 10.5 || 10.5
|}
 
PCR program:
* 95°C, 3 min.
* [95°C, 30 sec; 57°C, 30 sec.; 72°C, 30 sec.] x35 cycles
* 72°C, 3 min.
* 4°C ∞


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Revision as of 02:52, 14 November 2012

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November 6, 2012

  • Restriction digests of H2B-GFP and LOV
Restriction Digest Reagents
Reagent H2B-GFP/EcoRI H2B-GFP/PstI LOV/PstI
DNA(plasmid) 2.0 2.0 2.0
10x buffer 1.5 1.5 1.5
EcoRI 1.0 0 0
PstI 0 1.0 1.0
dH2O 10.5 10.5 10.5
Total 15.0 15.0 15.0


Expected:
1. H2B-GFP/ EcoRI = 4885, 210
2. H2B-GFP/ PstI = 5101
3. LOV/PstI = 251

Results of the digests are shown.

Results of the digests are shown.

  • ---Karmella 16:01, 7 November 2012 (EST): Note: Please re-do LOV/PstI


  • ---Karmella 16:01, 7 November 2012 (EST): Here is a reaction table for PCR
  • First, resuspend dry primer oligos (in blue-capped tubes) to 100 μM
  • Then make working solutions of 10 μM
  • Reactions:
  1. H2B-GFP plasmid + BB_H2B fwd + BB_H2B rev
  2. LOV plasmid + BB_LOV fwd + BB_LOV rev
Reagents H2B LOV
Plasmid DNA 0.5 μL 0.5
primer 1 (10 μM) 1.0 1.0
primer 2 (10 μM) 1.0 1.0
2x GoTaq mix 12.5 12.5
dH2O 10.5 10.5

PCR program:

  • 95°C, 3 min.
  • [95°C, 30 sec; 57°C, 30 sec.; 72°C, 30 sec.] x35 cycles
  • 72°C, 3 min.
  • 4°C ∞


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