Haynes Lab:Notebook/Investigating Photo-Switchable Synthetic Nucleosomes/2012/11/02: Difference between revisions

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{| class="wikitable" border="0" cellspacing="3" <!-- Digest check rxn. table -->
{| class="wikitable" border="0" cellspacing="3" <!-- Digest check rxn. table -->
|-valign="top"
|-valign="top"
| <u>Reagent</u> || <u>Volume</u>  
| <u>Reagent</u> || <u>LOV</u> || <u>H2B-GFP/ PstI</u> || <u>H2B-GFP/ EcoRI</u>
| rowspan="7" | <u>Expected:</u><br>1. LOV = 251, 3639<br>2. H2B-GFP/ PstI = 4885, 205<br>3. H2B-GFP/ EcoRI = ?
| rowspan="7" | <u>Expected:</u><br>1. LOV = 251, 3639<br>2. H2B-GFP/ PstI = 4885, 205<br>3. H2B-GFP/ EcoRI = ?
| rowspan="7" | [[Image:Vi110212_gel1.tif|150px|Digest check 11/02/12]]<br>15 μL/lane; 1% agarose
| rowspan="7" | [[Image:Vi110212_gel1.tif|150px|Digest check 11/02/12]]<br>15 μL/lane; 1% agarose
|-
| Reagent || LOV || H2B-GFP/ PstI || H2B-GFP/ EcoRI
|-
|-
| DNA(plasmid) || 2.0 μL || 2.0 || 2.0
| DNA(plasmid) || 2.0 μL || 2.0 || 2.0

Revision as of 08:45, 2 November 2012

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November 2, 2012 Digestion and PCR

  • Check LOV and H2B-GFP minipreps with digestion
  • BioBrick PCR

Minipreps

Measure concentrations

Sample 260/280 ng/μL
LOV 1.834 281.868
H2B-GFP 1.87 118.217

Check miniprep results

  • Use a plasmid editor software to check sequences for restriction digest enzymes
  • LOV has two PstI sites while H2B-GFP has one PstI, one NotI, and two EcoRI sites
  • Decide to cut LOV with PstI and H2B-GFP with PstI and EcoRI
  • Expected bands for LOV at 251 with PstI digest
  • Expected bands for H2B-GFP at 4885, 205 with Psti and EcoRI digest


Reagent LOV H2B-GFP/ PstI H2B-GFP/ EcoRI Expected:
1. LOV = 251, 3639
2. H2B-GFP/ PstI = 4885, 205
3. H2B-GFP/ EcoRI = ? 		Digest check 11/02/12
15 μL/lane; 1% agarose
DNA(plasmid) 2.0 μL 2.0 2.0
10X buffer 1.5 1.5 1.5 1.5
PstI 1.0 1.0 ---
EcoRI --- --- 1.0
dH2O 10.5 10.5 10.5
  15.0 15.0 15.0



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