User:Karmella Haynes/Notebook/BioBrick cloning/2015/04/21: Difference between revisions
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| || Rxn 1-4 || Neg | | || Rxn 1-4 || Neg | ||
|- | |- | ||
| Insert DNA || 3.0 || --- | | Insert DNA || 3.0 || --- | ||
|- | |- | ||
| Vector DNA (25 ng) || 3.5 || 3.5 | | Vector DNA (25 ng) || 3.5 || 3.5 | ||
|- | |- | ||
| 2x lgn buf (Roche) || 7.5 || 7.5 | | 2x lgn buf (Roche) || 7.5 || 7.5 | ||
|- | |- | ||
| T4 ligase (NEB) || 1.0 || 1.0 | | T4 ligase (NEB) || 1.0 || 1.0 | ||
|- | |- | ||
| dH<sub>2</sub>O || --- || 3.0 | | dH<sub>2</sub>O || --- || 3.0 | ||
|- | |- | ||
| || 15 μL || 15 μL | | || 15 μL || 15 μL | ||
|} | |} | ||
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| bgcolor=#cfcfcf | Mix(x13) | | bgcolor=#cfcfcf | Mix(x13) | ||
| rowspan=7 | Expected<br>pX330 = ~8335, 195<br>gRNA+pX330 = 8530 | | rowspan=7 | Expected<br>pX330 = ~8335, 195<br>gRNA+pX330 = 8530 | ||
| rowspan=7 | [[Image: | | rowspan=7 | [[Image:KAH042115_gel1.jpg|250px|Hover label]]<br>15 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] | ||
|- | |- | ||
| DNA || 3.0 || --- | | DNA || 3.0 || --- | ||
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Sequencing list: | Sequencing list: | ||
# g51-1, f | # g51-1, f (P139) | ||
# g51-1, r | # g51-1, r (P140) | ||
# | # g51-2, f | ||
# g51-2, r | |||
# g52-1, f | |||
# g52-1, r | |||
# g52-2, f | |||
# g52-2, r | |||
# g53-1, f | |||
# g53-1, r | |||
# g55-1, f | |||
# g55-1, r | |||
# g55-2, f | |||
# g55-2, r | |||
# g56-1, f | |||
# g56-1, r | |||
# g56-2, f | |||
# g56-2, r | |||
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| bgcolor=#cfcfcf | Rxn1,2 | | bgcolor=#cfcfcf | Rxn1,2 | ||
| bgcolor=#cfcfcf | Rxn3 | | bgcolor=#cfcfcf | Rxn3 | ||
| rowspan="7" | <u>Expected:</u><br>1,2. MV10 = 5191 | | rowspan="7" | <u>Expected:</u><br>1,2. MV10 = 5191<br>3. No product | ||
| rowspan="7" | [[Image: | | rowspan="7" | [[Image:KAH042215_gel1.jpg|250px|Hover name]]<br>5 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] | ||
|- | |- | ||
| DNA(clean linear) || 1.0 μL || 1.0 μL | | DNA(clean linear) || 1.0 μL || 1.0 μL | ||
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Conclusion | Conclusion | ||
* | * Lane 3 shows product. Must have accidentally added Phusion pol. | ||
* Desired band not any brighter than reaction done at 60°C (see [http://openwetware.org/wiki/User:Karmella_Haynes/Notebook/BioBrick_cloning/2015/04/17 04/17/15]). | |||
* Use reactions from 4/17/15 instead of these for LCR. | |||
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| bgcolor=#cfcfcf | Rxn1-4 | | bgcolor=#cfcfcf | Rxn1-4 | ||
| bgcolor=#cfcfcf | Rxn5,6 | | bgcolor=#cfcfcf | Rxn5,6 | ||
| rowspan="7" | <u>Expected:</u><br> | | rowspan="7" | <u>Expected:</u><br>5,6. Gal4DB-mCh = 1170<br>7-10. ATF2 = 906 | ||
| rowspan="7" | [[Image: | | rowspan="7" | [[Image:KAH042215_gel1.jpg|250px|Hover name]]<br>5 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] | ||
|- | |- | ||
| DNA(clean linear) || 0.5 μL || 2.0 μL | | DNA(clean linear) || 0.5 μL || 2.0 μL | ||
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Conclusion | Conclusion | ||
* | * Gal4DB-mCh - much better than previous reactions. Purify these (combined) and use for LCR | ||
* ATF2 - rxns w/ plasmid template look awesome! cDNA templates give a spurious band. Use plasmid PCR's (combined, purified) for LCR. | |||
Revision as of 15:40, 22 April 2015
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> | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
04/21/15
Ryan - Receiver plasmids, assembly
RESULTS (4/22/15)
Rene - check gRNA assemblies
LCR Development
Phusion PCR:
Program: Phusion (block A)
Program: Phusion (block B)
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