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 | |||
* LCR Development - assembly attempt #1 | |||
* Ryan - streak plates & liquid cultures (see 4/21/15) | * Ryan - streak plates & liquid cultures (see 4/21/15) | ||
---- | |||
'''Rene - minipreps & sequencing''' | |||
# gRNA53-2 | |||
# gRNA54 | |||
* Sigma GenElute kit | |||
* Elute w/ 75 μL elution sln. | |||
* Measure conc.'s | |||
{| {{table}} cellspacing="3" <!-- [DNA] table --> | |||
|- bgcolor=#cfcfcf | |||
| Sample || OD260 || 260/280 || ng/μL | |||
|- | |||
| 1. g53-2 || 0.186 || 1.863 || 186.1 | |||
|- | |||
| 2. g54 || 0.205 || 1.895 || 205.2 | |||
|} | |||
* Submit to DNASU for sequencing | |||
** Order Number is: 10068 (4/23/15) | |||
# g53-2, f (P139) - Confirmed | |||
# g53-2, r (P139) - low Phred 20 score | |||
# g54, f (P139) - Confirmed | |||
# g54, r (P139) - low Phred 20 score | |||
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| Sample || OD260 || 260/280 || ng/μL | | Sample || OD260 || 260/280 || ng/μL | ||
|- | |- | ||
| 1. 5'p Gal4DB-mCh PCR || | | 1. 5'p Gal4DB-mCh PCR || 0.139 || 1.825 || 138.6 | ||
|- | |- | ||
| 2. 5'p ATF2 PCR || | | 2. 5'p ATF2 PCR || 0.191 || 1.828 || 190.8 | ||
|- | |- | ||
| 2. 5'p MV10 PCR || | | 2. 5'p MV10 PCR || 0.066 || 1.716 || 65.9 | ||
|} | |} | ||
--- | Dilute the purified dsDNA to 30 fmol/μL (30 nM) | ||
* Formula: x μL = length in bp ÷ measured ng/μL * 0.0195 ng/μL * 50μL | |||
* | ** Gal4DB-mCh (1170 bp) = 8.2 | ||
** ATF2 (906 bp) = 4.6 | |||
** 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 | |||
* Use 1.0 μL of diluted dsDNA per 10 μL LCR reaction | |||
Prep the Oligo Bridges | |||
* Note: Final conc. in LCR rxn. is 30 nM each | |||
* Bring the IDT oligo pellet to 100μM with dH2O. nmoles oligo (on label) * 10 = μL H2O to add | |||
* Make a 300 nM working solution (final volume = 100 μL) in a new tube. 3 μL of 100μM oligo stock + 97 μL dH2O = 100 μL | |||
LCR Reactions (PCR fragments / oligo bridges) | |||
# 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 | |||
* | * Note: use '''5.0 μL of 15 fmol/μL MV10''' instead of 2.5 | ||
{| {{table}} cellspacing="3" | {| {{table}} cellspacing="3" | ||
|- | |- bgcolor=#cfcfcf | ||
| Reagent || Rxn1 || Rxn2 (neg) | |||
| | |||
|- | |- | ||
| DNA ( | | 30 nM DNA (3 nM) || 10.0 || 5.0 | ||
|- | |- | ||
| | | 300 nM Oligo Bridges (30 nM) || 7.5 || 7.5 | ||
|- | |- | ||
| | | 10X Ampligase Buffer || 2.5 || 2.5 | ||
|- | |- | ||
| | | Ampligase || 1.0 || 1.0 | ||
|- | |||
| dH<sub>2</sub>O || 4.0 || 6.5 | |||
|- | |- | ||
| || 25.0 | |||
| || | |||
|} | |} | ||
Thermocycler: BioRad dual block | |||
* 94 °C, 2 min. | |||
* 50x[94°C, 10 sec; 55°C, 30 sec; 66°C, 60 sec | |||
* 4 °C, ∞ | |||
Transformation | |||
* Add to 60 μL DH5α-turbo; incubate 5 min/ice | |||
* Plate on 100 μg/mL amp | |||
* Grow at 37°C overnight | |||
---- | |||
'''Ryan - Receiver cloning confirmations'''<br> | |||
* | * Check with E/X digests | ||
{| {{table}} cellspacing="3" <!-- | {| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table --> | ||
| | |- valign="top" | ||
| bgcolor=#cfcfcf | Reagent | |||
| bgcolor=#cfcfcf | Volume | |||
| 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" | [[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) || 3.0 μL | ||
|- | |- | ||
| | | 10X buffer || 1.5 | ||
|- | |- | ||
| | | EcoRI || 1.0 | ||
|- | |- | ||
| | | XbaI || 1.0 | ||
|- | |- | ||
| dH<sub>2</sub>O | | dH<sub>2</sub>O || 8.5 | ||
|- | |- | ||
| | | || 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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