Difference between revisions of "Haynes Lab:Notebook/Investigating Photo-Switchable Synthetic Nucleosomes/2013/02/08"

From OpenWetWare
Jump to: navigation, search
(Autocreate 2013/02/08 Entry for Haynes_Lab:Notebook/Investigating_Photo-Switchable_Synthetic_Nucleosomes)
 
(Entry title)
Line 6: Line 6:
 
| colspan="2"|
 
| colspan="2"|
 
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
 
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
==Entry title==
+
==February 8, 2013==
* Insert content here...
+
'''Type IIS Assembly
 +
* Attempt new assembly method because piecewise assembly was unsuccessful
 +
* Final assembly product: pSB1A3 left end / H2B / LOV +His tag/ pSB1A3 right end
 +
* Primer design
 +
H2B
 +
1A3/H2B fwd 5'-cacaccaCGTCTCa TAGA ATGCCAGAGCCAGCG
 +
H2B/LOV rev 5'-cacaccaCGTCTCa CCAA CTTAGCGCTGGTGTA
 +
 
 +
LOV
 +
LOV fwd 5'-cacaccaCGTCTCa TTGGCTACTACACTT
 +
LOV+his/1A3 rev 5'-cacaccaCGTCTCa TAGT ttagtggtgatggtgatgatgAAGTTCTTTTGCCGC
 +
 
 +
* First, resuspend dry primer oligos (in blue-capped tubes) to 100 μM
 +
* Then make working solutions of 10 μM
 +
* Reactions:
 +
 
 +
# H2B-GFP plasmid + 1A3/H2B fwd + H2B/LOV rev
 +
# LOV plasmid + LOV fwd + LOV+his/1A3 rev
 +
 
 +
{| class="wikitable" border=1 cellpadding="5" cellspacing="0"
 +
|-
 +
| Reagents || H2B || LOV
 +
|-
 +
| Plasmid DNA || 0.2 μL || 0.2
 +
|-
 +
| primer 1 (10 μM) || 1.0 || 1.0
 +
|-
 +
| primer 2 (10 μM) || 1.0 || 1.0
 +
|-
 +
| 2x GoTaq mix || 25.0 || 25.0
 +
|-
 +
| dH<sub>2</sub>O || 22.8 || 22.8
 +
|-
 +
| Total || 50.0 || 50.0
 +
|}
 +
 
 +
PCR program:
 +
* 95°C, 3 min.
 +
* [95°C, 30 sec; 57°C, 30 sec.; 72°C, 1 min.] x35 cycles
 +
* 72°C, 3 min.
 +
* 4°C ∞
 +
 
 +
{| class="wikitable" border=0
 +
|-
 +
| <u>Expected:</u><br>1. H2B = 410 <br>2. LOV = 461
 +
| <br>
 +
|}
 +
 
 +
<center>
 +
[[Image:Gel_2-8-13.png|Results of the digests are shown.]]
 +
[[Image:KAH_Fermentas_GeneRuler_1kbplus.jpg]]
 +
</center>
 +
 
 +
<center>
 +
Results of the PCR product digests are shown, with H2B on the left and LOV on the right. The gel was loaded with 5 μL samples.
 +
</center>
 +
 
 +
* The PCR products were purified with the Zymo DNA Clean & Concentrator Kit and eluted with 20 μL dH<sub>2</sub>O
 +
 
 +
{| class="wikitable" border=1 cellpadding="5" cellspacing="0"
 +
|-
 +
| PCR Product || 260|| 280 || 260/280 || ng/μL
 +
|-
 +
| H2B || fill || fill || fill || fill
 +
|-
 +
| LOV || fill || fill || fill || fill
 +
|}
  
  

Revision as of 04:39, 8 February 2013

Owwnotebook icon.png Project name <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

February 8, 2013

Type IIS Assembly

  • Attempt new assembly method because piecewise assembly was unsuccessful
  • Final assembly product: pSB1A3 left end / H2B / LOV +His tag/ pSB1A3 right end
  • Primer design

H2B 1A3/H2B fwd 5'-cacaccaCGTCTCa TAGA ATGCCAGAGCCAGCG H2B/LOV rev 5'-cacaccaCGTCTCa CCAA CTTAGCGCTGGTGTA

LOV LOV fwd 5'-cacaccaCGTCTCa TTGGCTACTACACTT LOV+his/1A3 rev 5'-cacaccaCGTCTCa TAGT ttagtggtgatggtgatgatgAAGTTCTTTTGCCGC

  • First, resuspend dry primer oligos (in blue-capped tubes) to 100 μM
  • Then make working solutions of 10 μM
  • Reactions:
  1. H2B-GFP plasmid + 1A3/H2B fwd + H2B/LOV rev
  2. LOV plasmid + LOV fwd + LOV+his/1A3 rev
Reagents H2B LOV
Plasmid DNA 0.2 μL 0.2
primer 1 (10 μM) 1.0 1.0
primer 2 (10 μM) 1.0 1.0
2x GoTaq mix 25.0 25.0
dH2O 22.8 22.8
Total 50.0 50.0

PCR program:

  • 95°C, 3 min.
  • [95°C, 30 sec; 57°C, 30 sec.; 72°C, 1 min.] x35 cycles
  • 72°C, 3 min.
  • 4°C ∞
Expected:
1. H2B = 410
2. LOV = 461

Results of the digests are shown. KAH Fermentas GeneRuler 1kbplus.jpg

Results of the PCR product digests are shown, with H2B on the left and LOV on the right. The gel was loaded with 5 μL samples.

  • The PCR products were purified with the Zymo DNA Clean & Concentrator Kit and eluted with 20 μL dH2O
PCR Product 260 280 260/280 ng/μL
H2B fill fill fill fill
LOV fill fill fill fill