User:Karmella Haynes/Notebook/BioBrick cloning/2015/04/22: Difference between revisions
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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 38.4 to make 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 |
Revision as of 17:35, 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> | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
04/22/15
Rene - minipreps & sequencing
LCR - assembly attempt #1
LCR Reactions (PCR fragments / oligo bridges)
Thermocycler: BioRad dual block
Minipreps
Assemblies
Oligo annealing
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