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

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==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 05:40, 8 February 2013

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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

Results of the digests are shown.

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



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