User:Karmella Haynes/Notebook/BioBrick cloning/2015/04/23: Difference between revisions
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== | ==04/23/15== | ||
<!-- Precede finished items with a checkmark ✓ --> | <!-- Precede finished items with a checkmark ✓ --> | ||
* | * Ryan - Receiver cloning stage 2 | ||
---- | ---- | ||
''' | '''Ryan - Receiver plasmids, assembly''' | ||
* | * Stage 1 - insert Regulator ORFS | ||
** Phusion PCR-amplify ORFs (add 5'-E, 3'-X cut sites) | |||
** Insert ORF(E/X) into Vector (E/X). see [https://benchling.com/s/VFNzuauP/edit Vector plasmid map] | |||
* Stage 2 - insert Promoters | |||
** '''Cut & dephos promoters with E/X''' | |||
** '''Insert promoters(E/X) into Vector/Regulator (BbsI) via Lig/Dig cycling''' | |||
{| {{table}} | |||
|- | '''Assemblies''' | ||
# pAub/AubR/MRV: pAub(E/Xdp)/153 + AubR/MRV(BbsI)/4087 | |||
| | # pBja/BjaR/MRV: pBja(E/Xdp)/122 + BjaR/MRV(BbsI)/3977 | ||
| | # pBra/BraR/MRV: pBra(E/Xdp)/214 + BraR/MRV(BbsI)/3977 | ||
# pRpa/RpaR/MRV: pRpa(E/Xdp)/136 + RpaR/MRV(BbsI)/3980 | |||
* Measure conc.'s | |||
{| {{table}} cellspacing="3" <!-- [DNA] table --> | |||
|- bgcolor=#cfcfcf | |||
| Sample || OD260 || 260/280 || ng/μL | |||
|- | |- | ||
| | | 1. AubR/MRV || 0.258 || 1.906 || 257.7 | ||
|- | |- | ||
| | | 2. BjaR/MRV || 0.177 || 1.904 || 177.3 | ||
|- | |- | ||
| | | 3. RpaR/MRV || 0.246 || 1.896 || 246.2 | ||
| | |||
| | |||
|} | |} | ||
* Digests (Fermentas FD) | * Digests (Fermentas FD) | ||
** | ** E/X | ||
** Inserts are gBlocks = 2.0 ng/μL | |||
{| {{table}} cellspacing="3" <!-- Digest rxn. table --> | {| {{table}} cellspacing="3" <!-- Digest for dephos rxn. table --> | ||
|- valign="top" | |- valign="top" | ||
| bgcolor=#cfcfcf | Reagent | | bgcolor=#cfcfcf | Reagent | ||
| bgcolor=#cfcfcf | Volume | | bgcolor=#cfcfcf | Volume | ||
|- | |- | ||
| DNA ( | | DNA (20 ng) || 10.0 μL | ||
|- | |- | ||
| 10x buffer || | | 10x buffer || 2.0 | ||
|- | |- | ||
| | | EcoRI || 1.0 | ||
|- | |- | ||
| | | XbaI || 1.0 | ||
|- | |- | ||
| | | dH<sub>2</sub>O || 6.0 | ||
|- | |- | ||
| | | || 20.0 μL --> 37°C/ ~15 min. | ||
|} | |} | ||
* Dephosphorylation (Roche) | * Dephosphorylation (Roche) | ||
** Assuming that shrimp alkaline phosphatase works in FastDigest buffer conditions [https://www.lifetechnologies.com/us/en/home/brands/thermo-scientific/molecular-biology/thermo-scientific-restriction-modifying-enzymes/modifying-enzymes-thermo-scientific/modifying-enzyme-buffers.html Link] | |||
{| {{table}} cellspacing="3" <!-- Dephos table --> | {| {{table}} cellspacing="3" <!-- Dephos table --> | ||
|- | |- | ||
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| bgcolor=#cfcfcf | Volume | | bgcolor=#cfcfcf | Volume | ||
|- | |- | ||
| DNA ( | | DNA (digest) || 20.0 μL (20 ng) | ||
|- | |- | ||
| phosphatase || 1.0 | | phosphatase || 1.0 | ||
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| dH<sub>2</sub>O || --- | | dH<sub>2</sub>O || --- | ||
|- | |- | ||
| || 20 | | || 20 μL | ||
|} | |} | ||
--> 37°C/ 10 min.; 75°C/ 2 min.; [final] = ~1 ng/μL | |||
* Digestion/Ligation Reactions (borrowed from Rene's gRNA/Cas9 assembly procedure) | |||
** Inserts are gBlocks = ~2ng/μL | |||
** For an insert = ~200 and vector = ~4000, need 5 ng insert for 2:1 insert:vector(50 ng) ratio | |||
# pAub/AubR/MRV: pAub(E/Xdp)/153 + AubR/MRV(BbsI)/4087 | |||
# pBja/BjaR/MRV: pBja(E/Xdp)/122 + BjaR/MRV(BbsI)/3977 | |||
# pRpa/RpaR/MRV: pRpa(E/Xdp)/136 + RpaR/MRV(BbsI)/3980 | |||
* Note: BraR cloning was not successful, omit from this round of assembly | |||
{| {{table}} cellspacing="3" <!-- Lig/Dig rxn table --> | |||
{| {{table}} cellspacing="3" <!-- | | || 1 || 2 || 3 | ||
| | |||
| | |||
|- | |- | ||
| | | Insert DNA || 5.0 || 5.0 || 5.0 | ||
|- | |- | ||
| 2. | | Vector DNA || 0.2 || 0.3 || 0.2 | ||
|- | |||
| 10x FD buf || 2.0 || 2.0 || 2.0 | |||
| | |||
|- | |- | ||
| | | 10 mM DTT || 1.0 || 1.0 || 1.0 | ||
|- | |- | ||
| | | 10 mM ATP || 1.0 || 1.0 || 1.0 | ||
|- | |- | ||
| | | FD BbsI/BpiI || 1.0 || 1.0 || 1.0 | ||
|- | |- | ||
| T4 ligase ( | | T4 ligase (Roche) || 1.0 || 1.0 || 1.0 | ||
|- | |- | ||
| dH<sub>2</sub>O || | | dH<sub>2</sub>O || 8.8 || 8.7 || 8.8 | ||
|- | |- | ||
| || | | || 20.0 μL || 20.0 μL || 20.0 μL || | ||
|} | |} | ||
- | Thermal cycler: Labnet - BbsI Dig/Lig | ||
* 6x[37°C, 5 min.; 23°C 5 min.] | |||
* 4°C, ∞ | |||
Revision as of 18:02, 23 April 2015
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04/23/15
Ryan - Receiver plasmids, assembly
--> 37°C/ 10 min.; 75°C/ 2 min.; [final] = ~1 ng/μL
Thermal cycler: Labnet - BbsI Dig/Lig
|