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

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(Autocreate 2013/02/08 Entry for Haynes_Lab:Notebook/Investigating_Photo-Switchable_Synthetic_Nucleosomes)
Current revision (08:52, 18 April 2013) (view source)
(February 8, 2013)
 
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==Entry title==
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==February 8, 2013==
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* Insert content here...
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'''Type IIS Assembly - Making PCR Products '''
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* Attempt new assembly method because piecewise assembly was unsuccessful
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* Final assembly product: pSB1A3 left end / H2B / LOV +His tag/ pSB1A3 right end
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* Primer design
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#H2B
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1A3/H2B fwd 5'-cacaccaCGTCTCa TAGA ATGCCAGAGCCAGCG
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H2B/LOV rev 5'-cacaccaCGTCTCa CCAA CTTAGCGCTGGTGTA
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#LOV
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LOV fwd 5'-cacaccaCGTCTCa TTGGCTACTACACTT
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LOV+his/1A3 rev 5'-cacaccaCGTCTCa TAGT ttagtggtgatggtgatgatgAAGTTCTTTTGCCGC
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* First, resuspend dry primer oligos (in blue-capped tubes) to 100 μM
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* Then make working solutions of 10 μM
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* Reactions:
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# H2B-GFP plasmid + 1A3/H2B fwd + H2B/LOV rev
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# LOV plasmid + LOV fwd + LOV+his/1A3 rev
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{| class="wikitable" border=1 cellpadding="5" cellspacing="0"
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|-
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| Reagents || H2B || LOV
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|-
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| Plasmid DNA || 0.2 μL || 0.2
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|-
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| primer 1 (10 μM) || 1.0 || 1.0
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|-
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| primer 2 (10 μM) || 1.0 || 1.0
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|-
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| 2x GoTaq mix || 25.0 || 25.0
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|-
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| dH<sub>2</sub>O || 22.8 || 22.8
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|-
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| Total || 50.0 || 50.0
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|}
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PCR program:
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* 95°C, 3 min.
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* [95°C, 30 sec; 57°C, 30 sec.; 72°C, 1 min.] x35 cycles
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* 72°C, 3 min.
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* 4°C ∞
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{| class="wikitable" border=0
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|-
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| <u>Expected:</u><br>1. H2B = 410 <br>2. LOV = 461
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| <br>
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|}
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<center>
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[[Image:VN_Gel_2-8-13.png|Results of the digests are shown.]]
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[[Image:KAH_Fermentas_GeneRuler_1kbplus.jpg]]
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</center>
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<center>
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Results of the PCR product digests are shown.  It was later found that the transparency used was blocking the UV light, so no bands could be seen.
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</center>
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* The PCR products were purified with the Zymo DNA Clean & Concentrator Kit and eluted with 20 μL dH<sub>2</sub>O
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{| class="wikitable" border=1 cellpadding="5" cellspacing="0"
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|-
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| PCR Product || 260|| 280 || 260/280 || ng/μL
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|-
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| H2B || 0.078|| 0.043|| 1.81 || 77.995
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|-
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| LOV || 0.08 || 0.044 || 1.817 || 80.443
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|-
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| pSB1A3 || 0.023 || 0.014 || 1.727 || 23.386
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|-
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|}

Current revision

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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. Image:KAH_Fermentas_GeneRuler_1kbplus.jpg

Results of the PCR product digests are shown. It was later found that the transparency used was blocking the UV light, so no bands could be seen.

  • 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 0.078 0.043 1.81 77.995
LOV 0.08 0.044 1.817 80.443
pSB1A3 0.023 0.014 1.727 23.386



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