Haynes Lab:Notebook/Investigating Photo-Switchable Synthetic Nucleosomes/2013/02/08

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(Autocreate 2013/02/08 Entry for Haynes_Lab:Notebook/Investigating_Photo-Switchable_Synthetic_Nucleosomes)
(February 8, 2013)
(2 intermediate revisions not shown.)
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 - Making PCR Products '''
 +
* 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 08:40, 8 February 2013

Project name Main project page
Previous entry      Next entry

February 8, 2013

Type IIS Assembly - Making PCR Products

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

1A3/H2B fwd 5'-cacaccaCGTCTCa TAGA ATGCCAGAGCCAGCG

H2B/LOV rev 5'-cacaccaCGTCTCa CCAA CTTAGCGCTGGTGTA

  1. 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

Image:Gel 2-8-13.png Image: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



Personal tools