User:Karmella Haynes/Notebook/BioBrick cloning/2015/05/05: Difference between revisions
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| bgcolor=#cfcfcf | Volume | | bgcolor=#cfcfcf | Volume | ||
| rowspan="7" | <u>Expected:</u><br>KAH87 fwd/MV10 = '''~6150''', 36, 28<br>KAH87 rev/MV10 = ~5100, '''1089''', 28<br>MV10 = ~5100, 36, 28<br> | | rowspan="7" | <u>Expected:</u><br>KAH87 fwd/MV10 = '''~6150''', 36, 28<br>KAH87 rev/MV10 = ~5100, '''1089''', 28<br>MV10 = ~5100, 36, 28<br> | ||
| rowspan="7" | [[Image: | | rowspan="7" | [[Image:Somegel.jpg|400px|Hover name]]<br>15 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] | ||
|- | |- | ||
| DNA(plasmid) || 2.0 μL | | DNA(plasmid) || 2.0 μL | ||
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| || 15 μL --> 37°C/ ~15 min. | | || 15 μL --> 37°C/ ~15 min. | ||
|} | |} | ||
* Measure conc.'s | |||
{| {{table}} cellspacing="3" <!-- [DNA] table --> | |||
|- bgcolor=#cfcfcf | |||
| Sample || OD260 || 260/280 || ng/μL | |||
|- | |||
| 1. KAH87/MV10-1 || 0.273 || 1.948 || 273.0 | |||
|- | |||
| 2. KAH87/MV10-2 || 0.341 || 1.940 || 341.5 | |||
|- | |||
| 3. '''KAH87/MV10-3''' || 0.319 || 1.937 || 318.5 | |||
|- | |||
| 4. KAH87/MV10-4 || 0.248 || 1.948 || 248.1 | |||
|} | |||
Conclusions: | |||
* Success! Clone #3 has forward insertion | |||
* Discard - clones 1, 2, 4 are reverse insertions | |||
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* Phusion PCR-amplify promoters | * Phusion PCR-amplify promoters | ||
# pAubR, EcoRI fwd, | # pAubR, EcoRI fwd, SpeI rev | ||
# pBjaR, EcoRI fwd, | # pBjaR, EcoRI fwd, SpeI rev | ||
# pBraR, EcoRI fwd, | # pBraR, EcoRI fwd, SpeI rev | ||
# pRpaR, EcoRI fwd, | # pRpaR, EcoRI fwd, SpeI rev | ||
{| {{table}} cellspacing="3" <!-- PCR rxn table --> | {| {{table}} cellspacing="3" <!-- PCR rxn table --> | ||
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Thermal cycler: Bio-Rad - Phusion | Thermal cycler: Bio-Rad - Phusion | ||
* 98°C 3 min. | * 98°C 3 min. | ||
* 30x[98°C, | * 30x[98°C, 10 sec; 66°C 30 sec; 72°C 30 sec] | ||
* 72°C 3 min. | * 72°C 3 min. | ||
* 4°C, ∞ | * 4°C, ∞ | ||
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** Qiagen PCR purification kit | ** Qiagen PCR purification kit | ||
** Elute w/ 30 μL elution buffer | ** Elute w/ 30 μL elution buffer | ||
* Measure conc.'s | |||
{| {{table}} cellspacing="3" <!-- [DNA] table --> | |||
|- bgcolor=#cfcfcf | |||
| Sample || OD260 || 260/280 || ng/μL | |||
|- | |||
| 1. pAubR PCR || 0.021 || 1.707 || 20.8 | |||
|- | |||
| 2. pBjaR PCR || 0.018 || 1.851 || 18.3 | |||
|- | |||
| 3. pBraR PCR || 0.026 || 1.807 || 25.5 | |||
|- | |||
| 4. pRpaR PCR || 0.018 || 1.720 || 17.8 | |||
|} | |||
* Digests (Fermentas FD) | |||
** Use clear FD buffer (no dye) | |||
{| {{table}} cellspacing="3" <!-- Digest rxn. table --> | |||
|- valign="top" | |||
| bgcolor=#cfcfcf | Reagent | |||
| bgcolor=#cfcfcf | Volume | |||
|- | |||
| DNA (250 ng PCR) || up to 16.0 | |||
|- | |||
| 10x clear FD buffer || 2.0 | |||
|- | |||
| EcoRI || 1.0 | |||
|- | |||
| SpeI || 1.0 | |||
|- | |||
| dH<sub>2</sub>O || --- | |||
|- | |||
| || 20.0 μL | |||
|} | |||
--> 37°C/ ~15 min. | |||
* Dephosphorylation (Roche) | |||
** Add 1.0 SAP (Roche) to each rxn. | |||
** 37°C/ 10 min.; 75°C/ 2 min.; [final] = 12.5 ng/μL | |||
* Dig/Lig reactions | |||
** 2:1 ratio calculation: ~150 bp insert / ~4000 bp vector * 2 * 100 = '''3.75 ng insert''' | |||
# pAub-PCR(E/S dp) + AubR/MRV | |||
# pBja-PCR(E/S dp) + BjaR/MRV | |||
# pBra-PCR(E/S dp) + BraR/MRV | |||
# pRpa-PCR(E/S dp) + RpaR/MRV | |||
{| {{table}} cellspacing="3" <!-- Oligo annealing table --> | |||
|- valign="top" | |||
| bgcolor=#cfcfcf | Reagent | |||
| bgcolor=#cfcfcf | Rxn | |||
| bgcolor=#cfcfcf | Mix (x5) | |||
|- | |||
| 100 ng Vector || ### (up to 13.5) || --- | |||
|- | |||
| insert || 1.0 || --- | |||
|- | |||
| 10x FD buf || 2.0 || 10.0 | |||
|- | |||
| 10 mM DTT || 1.0 || 5.0 | |||
|- | |||
| 10 mM ATP || 1.0 || 5.0 | |||
|- | |||
| FastDigest BbsI/BpiI || 1.0 || 5.0 | |||
|- | |||
| Roche T4 DNA ligase || 0.5 || 2.5 | |||
|- | |||
| dH<sub>2</sub>O || ### || --- | |||
|- | |||
| || 20.0 | |||
|} | |||
--> Pipette 5.5 μL master mix into each PCR tube<br> | |||
--> Add 1.0 μL insert into each tube<br> | |||
--> Add 100 ng vector DNA<br> | |||
--> Add 13.5 - vector volume dH<sub>2</sub>O | |||
--> Mix by flicking | |||
LabNet OptiMax Thermocycler: '''BbsI Dig/Lig''' | |||
* 6x [37°C, 5 min; 23°C, 5 min] | |||
* 4°C, ∞ | |||
Transformation(s) | |||
* Thaw 2 tubes chem competent DH5α-turbo on ice | |||
* Transfer 10.0 μL each ligation to fresh sterile 0.5 mL tube | |||
* Add 50 μL DH5α-turbo; pipette up-and-down 3x GENTLY | |||
* Incubate 5 min. on ice | |||
* Plate on 100 μg/mL amp | |||
** Split ligations in 1/2, do rapid protocol | |||
RESULTS (5/06/15) | |||
* tba | |||
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Revision as of 19:39, 5 May 2015
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05/05/15
Minipreps
Conclusions:
Ryan - PCR & Dig/Lig (promoter insert trial #3) Ryan - Receiver plasmids, assembly - REVISED STRATEGY (PCR promoters)
Thermal cycler: Bio-Rad - Phusion
--> 37°C/ ~15 min.
--> Pipette 5.5 μL master mix into each PCR tube LabNet OptiMax Thermocycler: BbsI Dig/Lig
RESULTS (5/06/15)
Cas-tone Project
Assemblies
Oligo annealing
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