User:Karmella Haynes/Notebook/BioBrick cloning/2015/04/21: Difference between revisions
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Karmella's BioBrick Cloning</span> | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Karmella's BioBrick Cloning</span> | ||
|style="background-color: #F2F2F2" align="center"| | |style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]] }}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}} | ||
|- | |- | ||
| colspan="2"| | | colspan="2"| | ||
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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 | ||
|} | |} | ||
* Transform 50 uL DH5α-turbo | * Transform 50 uL DH5α-turbo | ||
* Plate on 100 μg/mL amp | * Plate on 100 μg/mL amp | ||
RESULTS (4/22/15) | |||
# AubR/MRV: 11 colonies | |||
# BjaR/MRV: 11 colonies | |||
# BraR/MRV: 6 colonies | |||
# RpaR/MRV: 7 colonies | |||
# MRV (neg): 3 colonies | |||
* Success! Pick 2 colonies from plates 1-4 for streak plate and liquid cultures (5 mL each) | |||
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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) - Confirmed | ||
# g51-1, r | # g51-1, r (P140) - low Phred 20 score | ||
# | # g51-2, f - Confirmed | ||
# g51-2, r - low Phred 20 score | |||
# g52-1, f - Confirmed | |||
# g52-1, r - low Phred 20 score | |||
# g52-2, f - Confirmed | |||
# g52-2, r - low Phred 20 score | |||
# g53-1, f - Confirmed | |||
# g53-1, r - low Phred 20 score | |||
# g55-1, f - Confirmed | |||
# g55-1, r - low Phred 20 score | |||
# g55-2, f - Confirmed | |||
# g55-2, r - low Phred 20 score | |||
# g56-1, f - Confirmed | |||
# g56-1, r - low Phred 20 score | |||
# <font color="red">g56-2, f - questionable sequence across gRNA, discard | |||
# g56-2, r - low Phred 20 score</font> | |||
* Cultures for new minipreps | * Cultures for new minipreps | ||
** 6 & 7 were accidentally mixed | ** 6 & 7 were accidentally mixed - discard these | ||
** Set up 5 mL cultures for gRNA53-2 and gRNA54 | ** Set up 5 mL cultures for gRNA53-2 and gRNA54 | ||
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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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Program: Phusion (block B) | Program: Phusion (block B) | ||
* 98°C, 3 min | * 98°C, 3 min | ||
* 35x[98°C, 10 sec; | * 35x[98°C, 10 sec; 55°C, 30 sec; 72°C, 60 sec] | ||
* 72°C, 10 min | * 72°C, 10 min | ||
* 4°C ∞ | * 4°C ∞ | ||
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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. | |||
Latest revision as of 00:55, 27 September 2017
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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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