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

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(Entry title)
Current revision (04:52, 14 November 2012) (view source)
(November 6, 2012)
 
(22 intermediate revisions not shown.)
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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 ∞
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->

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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. Image:KAH_Fermentas_GeneRuler_1kbplus.jpg

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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