User:Karmella Haynes/Notebook/BioBrick cloning/2015/04/22: 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}}}}]]}} | ||
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==04/22/15== | ==04/22/15== | ||
<!-- Precede finished items with a checkmark ✓ --> | <!-- Precede finished items with a checkmark ✓ --> | ||
* Rene - minipreps & sequencing: g53-2, g54 | * Rene - minipreps & sequencing: g53-2, g54 | ||
* LCR Development - assembly attempt #1 | * LCR Development - assembly attempt #1 | ||
* Ryan - streak plates & liquid cultures (see 4/21/15) | |||
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* Submit to DNASU for sequencing | * Submit to DNASU for sequencing | ||
# g53-2, f (P139) | ** Order Number is: 10068 (4/23/15) | ||
# g53-2, r (P139) | # g53-2, f (P139) - Confirmed | ||
# g54, f (P139) | # g53-2, r (P139) - low Phred 20 score | ||
# g54, r (P139) | # g54, f (P139) - Confirmed | ||
# g54, r (P139) - low Phred 20 score | |||
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Dilute the purified dsDNA to 30 fmol/μL (30 nM) | Dilute the purified dsDNA to 30 fmol/μL (30 nM) | ||
* Formula: x μL = length in bp ÷ measured ng/μL * 0.0195 ng/μL * 50μL | * Formula: x μL = length in bp ÷ measured ng/μL * 0.0195 ng/μL * 50μL | ||
** Gal4DB-mCh (1170 bp) = | ** Gal4DB-mCh (1170 bp) = 8.2 | ||
** ATF2 (906 bp) = | ** ATF2 (906 bp) = 4.6 | ||
** MV10 (5191 bp) = | ** MV10 (5191 bp) = <font color=red>76.8</font> -- use 19.2 to make 25 uL of 15 fmol/μL sln., use 2x | ||
* Add x to dH<sub>2</sub>O, final vol. = 50 μL | * Add x to dH<sub>2</sub>O, final vol. = 50 μL | ||
* Use 1.0 μL of diluted dsDNA per 10 μL LCR reaction | * Use 1.0 μL of diluted dsDNA per 10 μL LCR reaction | ||
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# Gal4DB-mCh, ATF2, MV10 / LCRb_MV10_Gal4DB_rc, LCRb_mCh_ATF2_rc, LCRb_ATF2_MV10_rc | # Gal4DB-mCh, ATF2, MV10 / LCRb_MV10_Gal4DB_rc, LCRb_mCh_ATF2_rc, LCRb_ATF2_MV10_rc | ||
# Neg: MV10 / LCRb_MV10_Gal4DB_rc, LCRb_mCh_ATF2_rc, LCRb_ATF2_MV10_rc | # Neg: MV10 / LCRb_MV10_Gal4DB_rc, LCRb_mCh_ATF2_rc, LCRb_ATF2_MV10_rc | ||
* Note: use '''5.0 μL of 15 fmol/μL MV10''' instead of 2.5 | |||
{| {{table}} cellspacing="3" | {| {{table}} cellspacing="3" | ||
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| Reagent || Rxn1 || Rxn2 (neg) | | Reagent || Rxn1 || Rxn2 (neg) | ||
|- | |- | ||
| 30 nM DNA (3 nM) || | | 30 nM DNA (3 nM) || 10.0 || 5.0 | ||
|- | |- | ||
| 300 nM Oligo Bridges (30 nM) || 7.5 || 7.5 | | 300 nM Oligo Bridges (30 nM) || 7.5 || 7.5 | ||
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| Ampligase || 1.0 || 1.0 | | Ampligase || 1.0 || 1.0 | ||
|- | |- | ||
| dH<sub>2</sub>O || | | dH<sub>2</sub>O || 4.0 || 6.5 | ||
|- | |- | ||
| || 25.0 | | || 25.0 | ||
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---- | ---- | ||
''' | '''Ryan - Receiver cloning confirmations'''<br> | ||
* Check with E/ | |||
* Check with E/X digests | |||
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table --> | {| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table --> | ||
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| bgcolor=#cfcfcf | Reagent | | bgcolor=#cfcfcf | Reagent | ||
| bgcolor=#cfcfcf | Volume | | bgcolor=#cfcfcf | Volume | ||
| rowspan="7" | <u>Expected:</u><br>1. | | rowspan="7" | <u>Expected:</u><br>1,2. AubR/MRV = ~3200, 886<br>3,4. BjaR/MRV = ~3200, 776<br>5,6. BraR/MRV = ~3200, 776<br>7,8. RpaR/MRV = ~3200, 779<br>MRV (empty) = ~3200 | ||
| rowspan="7" | | | rowspan="7" | [[Image:KAH042315_gel1.jpg|250px|Hover name]]<br>15 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] | ||
|- | |- | ||
| DNA(plasmid) || | | DNA(plasmid) || 3.0 μL | ||
|- | |- | ||
| 10X buffer || 1.5 | | 10X buffer || 1.5 | ||
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| EcoRI || 1.0 | | EcoRI || 1.0 | ||
|- | |- | ||
| | | XbaI || 1.0 | ||
|- | |- | ||
| dH<sub>2</sub>O || | | dH<sub>2</sub>O || 8.5 | ||
|- | |- | ||
| || 15 μL --> 37°C/ ~15 min. | | || 15 μL --> 37°C/ ~15 min. | ||
|} | |} | ||
* | Conclusions | ||
* AubR/MRV - success! Use clone 1 (lane 1) | |||
* BjaR/MRV - success! Use clone 1 (lane 3) | |||
* BraR/MRV - failed. Pick more clones | |||
* RpaR/MRV - success! Use clone 2 (lane 8) | |||
Latest revision as of 00:55, 27 September 2017
Karmella's BioBrick Cloning | Main project page Previous entry Next entry | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
04/22/15
Rene - minipreps & sequencing
LCR - assembly attempt #1
LCR Reactions (PCR fragments / oligo bridges)
Thermocycler: BioRad dual block
Ryan - Receiver cloning confirmations
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