User:Karmella Haynes/Notebook/BioBrick cloning/2012/11/30: Difference between revisions
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(Autocreate 2012/11/30 Entry for User:Karmella_Haynes/Notebook/BioBrick_cloning) |
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== | ==11/30/12== | ||
<!-- Precede finished items with a checkmark ✓ --> | <!-- Precede finished items with a checkmark ✓ --> | ||
* | * Assembly: CMVTetO2:K (KAH3)/ MV; expression vector for Vi's mammalian cell expression experiment | ||
---- | ---- | ||
'''Assemblies''' | '''Assemblies''' | ||
# | # KAH3/MV2: (1) KAH3/(X/P)/751 + (2) MV2/(S/P)/4529 | ||
> Digests (Fermentas FD)<br> | > Digests (Fermentas FD)<br> | ||
{| class="wikitable" border="0" cellspacing="3" <!-- Digest rxn. table --> | {| class="wikitable" border="0" cellspacing="3" <!-- Digest rxn. table --> | ||
|-valign="top" | |-valign="top" | ||
| <u>Reagent</u> || <u>Volume</u> || | | <u>Reagent</u> || <u>Volume</u> || | ||
| rowspan="9" | | | rowspan="9" | [[Image:KAH113012_gel1.jpg |150px|Hover name]]<br>30 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] | ||
|- | |- | ||
| DNA (plasmid) || up to 25 μL | | DNA (plasmid) || up to 25 μL | ||
Line 58: | Line 33: | ||
| dH<sub>2</sub>O || --- | | dH<sub>2</sub>O || --- | ||
|- | |- | ||
| || 30 μL --> 37°C/ ~ | | || 30 μL --> 37°C/ ~15 min. | ||
|} | |} | ||
> Measure conc.'s | > Measure conc.'s of gel purified fragments | ||
{| class="wikitable" border="0" cellspacing="3" <!-- [DNA] table --> | {| class="wikitable" border="0" cellspacing="3" <!-- [DNA] table --> | ||
|- | |- | ||
| <u>Sample</u> || <u>OD260</u> || <u>260/280</u> || <u>ng/μL</u> | | <u>Sample</u> || <u>OD260</u> || <u>260/280</u> || <u>ng/μL</u> | ||
|- | |- | ||
| 1. | | 1. KAH3 (X/P) || 0.028 || 1.96 || 28.41 | ||
| | |||
|- | |- | ||
| | | 2. MV2 (S/P) || 0.085 || 1.93 || 85.02 | ||
|} | |} | ||
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> Ligations | > Ligations | ||
{| class="wikitable" border="0" cellspacing="3" <!-- Ligations table --> | {| class="wikitable" border="0" cellspacing="3" <!-- Ligations table --> | ||
| <u>Ligation</u> || <font color="blue"><u>Plate results (lig : neg crtl)</u> | | <u>Ligation</u> || <font color="blue"><u>Plate results (lig : neg crtl)</u> 11/31/12</font> | ||
|- | |- | ||
| 1. | | 1. KAH3 (X/P)/751, 8 ng + MV2 (S/P)/4529, 25 ng || <font color="blue">KAH3/MV2 >10:1 (Pick 2)</font> | ||
|- | |- | ||
| 2. | | 2. MV2 (S/P)/ 25 ng || zero colonies | ||
|} | |} | ||
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| || 1 || 2 || | | || 1 || 2 || | ||
|- | |- | ||
| Insert DNA || | | Insert DNA || 0.3 || --- || | ||
|- | |- | ||
| Vector DNA || | | Vector DNA || 0.3 || 0.3 || | ||
|- | |- | ||
| 2x lgn buf (Roche) || | | 2x lgn buf (Roche) || 5.0 || 5.0 || | ||
|- | |- | ||
| T4 ligase (NEB) || 1.0 || 1.0 || | | T4 ligase (NEB) || 1.0 || 1.0 || | ||
|- | |- | ||
| dH<sub>2</sub>O || | | dH<sub>2</sub>O || 3.4 || 3.7 || | ||
|- | |- | ||
| || | | || 10 μL || 10 μL || | ||
|} | |} | ||
- | * Follow quick transformation protocol | ||
* Add 30 uL DH5α-Turbo to each ligation; plate on 100 μg/mL Amp agar | |||
Revision as of 20:56, 19 March 2013
Karmella's BioBrick Cloning | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
11/30/12
Assemblies
> Digests (Fermentas FD)
|