User:Karmella Haynes/Notebook/BioBrick cloning/2015/04/23: Difference between revisions

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* Ryan - Receiver cloning stage 2
* Ryan - Receiver cloning stage 2
* Line item 2




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* Digests (Fermentas FD)
* Digests (Fermentas FD)
** E/X
** E/X
** Inserts are gBlocks = 2.0 ng/μL


{| {{table}} cellspacing="3" <!-- Digest for dephos rxn. table -->
{| {{table}} cellspacing="3" <!-- Digest for dephos rxn. table -->
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| bgcolor=#cfcfcf | Volume
| bgcolor=#cfcfcf | Volume
|-
|-
| DNA (500 ng plasmid) || up to 16.0 μL
| DNA (20 ng) || 10.0 μL
|-
|-
| 10x buffer || 2.0
| 10x buffer || 2.0
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| XbaI || 1.0
| XbaI || 1.0
|-
|-
| dH<sub>2</sub>O || ###
| dH<sub>2</sub>O || 6.0
|-
|-
| &nbsp; || 20.0 μL --> 37°C/ ~15 min.
| &nbsp; || 20.0 μL --> 37°C/ ~15 min.
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| bgcolor=#cfcfcf | Volume
| bgcolor=#cfcfcf | Volume
|-
|-
| DNA (digest) || 20.0 μL (500 ng)
| 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 || ---
|-
|-
| &nbsp; || 20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = ~25 ng/μL
| &nbsp; || 20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = ~1 ng/μL
|}
|}


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* Ligation/Digestion Reactions
* Ligation/Digestion Reactions
** For an insert = ~200 and vector = ~4000, need 5 ng insert for 2:1 insert:vector ratio
** Inserts are gBlocks = ~2ng/μL
** Insert is mostly old vector (3000 bp), ~6% is insert
** For an insert = ~200 and vector = ~4000, need 5 ng insert for 2:1 insert:vector(50 ng) ratio
** So, use at least 84 ng of the E/X dephos insert DNA
 


{| {{table}} cellspacing="3" <!-- Lig/Dig rxn table -->
{| {{table}} cellspacing="3" <!-- Lig/Dig rxn table -->
| &nbsp;            || 1    || 2    ||
| &nbsp;            || 1    || 2    ||
|-
|-
| Insert DNA        || ### || ---  ||
| Insert DNA        || 5.0 || ---  ||
|-
|-
| Vector DNA        || ###  || ###  ||
| Vector DNA        || ###  || ###  ||
|-
|-
| 2x lgn buf (Roche) || ### || ###  ||
| 2x lgn buf (Roche) || 7.5 || ###  ||
|-
|-
| T4 ligase (NEB)    || 1.0  || 1.0  ||
| T4 ligase (NEB)    || 1.0  || 1.0  ||
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| dH<sub>2</sub>O    || ###  || ###  ||
| dH<sub>2</sub>O    || ###  || ###  ||
|-
|-
| &nbsp;            || # μL || # μL ||
| &nbsp;            || 15.0 μL || # μL ||
|}
|}
Thermal cycler: Bio-Rad C1000
*




Revision as of 16:38, 23 April 2015

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04/23/15

  • 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 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


Assemblies

  1. pAub/AubR/MRV: pAub(E/Xdp)/153 + AubR/MRV(BbsI)/4087
  2. pBja/BjaR/MRV: pBja(E/Xdp)/122 + BjaR/MRV(BbsI)/3977
  3. pBra/BraR/MRV: pBra(E/Xdp)/214 + BraR/MRV(BbsI)/3977
  4. pRpa/RpaR/MRV: pRpa(E/Xdp)/136 + RpaR/MRV(BbsI)/3980


  • Measure conc.'s
Sample OD260 260/280 ng/μL
1. 5'p Gal4DB-mCh PCR 0.139 1.825 138.6
2. 5'p ATF2 PCR 0.191 1.828 190.8
3. 5'p MV10 PCR 0.066 1.716 65.9


  • Digests (Fermentas FD)
    • E/X
    • Inserts are gBlocks = 2.0 ng/μL
Reagent Volume
DNA (20 ng) 10.0 μL
10x buffer 2.0
EcoRI 1.0
XbaI 1.0
dH2O 6.0
  20.0 μL --> 37°C/ ~15 min.


  • Dephosphorylation (Roche)
    • Assuming that shrimp alkaline phosphatase works in FastDigest buffer conditions Link
Reagent Volume
DNA (digest) 20.0 μL (20 ng)
phosphatase 1.0
dH2O ---
  20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = ~1 ng/μL


  • Ligation/Digestion Reactions
    • Inserts are gBlocks = ~2ng/μL
    • For an insert = ~200 and vector = ~4000, need 5 ng insert for 2:1 insert:vector(50 ng) ratio


  1 2
Insert DNA 5.0 ---
Vector DNA ### ###
2x lgn buf (Roche) 7.5 ###
T4 ligase (NEB) 1.0 1.0
dH2O ### ###
  15.0 μL # μL

Thermal cycler: Bio-Rad C1000



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