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

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* Expected bands for LOV at 251 with PstI digest
* Expected bands for LOV at 251 with PstI digest
* Expected bands for H2B-GFP at 4885, 205 with Psti and EcoRI digest
* Expected bands for H2B-GFP at 4885, 205 with Psti and EcoRI digest
Digests'''<br>
> Mut mCh clones from last week<br>
> Check with BamHI/KpnI/PstI digests (plasmid = pJ10:G04381)
{| class="wikitable" border="0" cellspacing="3" <!-- Digest check rxn. table -->
|-valign="top"
| <u>Reagent</u> || <u>Volume</u>
| rowspan="7" | <u>Expected:</u><br>mut = 751, 3639<br>wt = 388, 363, 3639<br>
| rowspan="7" | [[Image:Vi110212_gel1.tif|150px|Digest check 11/02/12]]<br>15 μL/lane; 1% agarose
|-
| DNA(plasmid) || 2.0 μL
|-
| 10X buffer || 1.5
|-
| BamHI || 1.0
|-
| KpnI || 1.0
|-
| PstI || 1.0
|-
| dH<sub>2</sub>O || 8.5
|-
| &nbsp; || 15 μL --> 37°C/ ~15 min.
|}


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Revision as of 08:29, 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

Digests
 > Mut mCh clones from last week
> Check with BamHI/KpnI/PstI digests (plasmid = pJ10:G04381)

Reagent Volume Expected:
mut = 751, 3639
wt = 388, 363, 3639
 Digest check 11/02/12
15 μL/lane; 1% agarose
DNA(plasmid) 2.0 μL
10X buffer 1.5
BamHI 1.0
KpnI 1.0
PstI 1.0
dH2O 8.5
  15 μL --> 37°C/ ~15 min.



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