User:Karmella Haynes/Notebook/BioBrick cloning/2015/04/28: Difference between revisions

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| bgcolor=#cfcfcf | Vol
| bgcolor=#cfcfcf | Vol
| bgcolor=#cfcfcf | Mix (x2)
| bgcolor=#cfcfcf | Mix (x2)
| rowspan=7 || Expected:<br>1. pAubR = 153<br>2. pBjaR = 122<br>3. pBraR = 214<br>4. pRpaR = 136
| rowspan=7 | Expected:<br>1. pAubR = 153<br>2. pBjaR = 122<br>3. pBraR = 214<br>4. pRpaR = 136
|-
|-
| gBlock DNA (2 ng/μL) || 0.5  || 1.0  
| gBlock DNA (2 ng/μL) || 0.5  || 1.0  

Revision as of 18:11, 28 April 2015

Karmella's BioBrick Cloning <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
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04/28/15

  • Ryan - minipreps & digests
  • Ryan - PCR & Dig/Lig (promoter insert trial #2)


Minipreps

  • Use Sigma GenElute; elute with 75 μL elution solution
  • BraR/MRV colonies - picked 4 additional colonies from original cloning plate
  • Cut w/ E/X
  1. Bra/MRV 3
  2. Bra/MRV 4
  3. Bra/MRV 5
  4. Bra/MRV 6
  • promoter/Regulator/MRV colonies
  • Cut w/ KpnI/EcoRI - BbsI drop-in of promoter destroys KpnI site; empty vector will produce 2 bands, successful clones will be linearized (1 cut)
  1. pAub/AubR/MRV
  2. pBja/BjaR/MRV
  3. pRpa/RpaR/MRV


Reagent Volume Expected:
1-4. Bra/MRV = ~3200, 776
Empty MRV E/X = ~3200
5. pAub/AubR/MRV = 4240
6. pBja/BjaR/MRV = 4099
7. pRpa/RpaR/MRV = 4116
Empty vector = ~4000, 971
DNA(plasmid) 3.0 μL
10X buffer 1.5
enzyme 1 1.0
enzyme 2 1.0
dH2O 8.5
  15 μL --> 37°C/ ~15 min.


  • Measure [DNA]
Sample OD 260 260/280 ng/μL
BraR/MRV #5 0.465 1.913 465.4


Conclusions

  • BraR/MRV - Success! Keep colony 5 (lane 3)
  • promter/Reg/MRV - none worked. Try the assembly again with PCR-amplified promoter inserts


Ryan - PCR & Dig/Lig (promoter insert trial #2)

Ryan - Receiver plasmids, assembly - REVISED STRATEGY (PCR promoters)

  • 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
    • PCR-amplify promoter inserts (same primers as Stage 1)
    • 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
    • Values from last week, except BraR/MRV, which was measured today
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. BraR/MRV #5 0.465 1.913 465.4
4. RpaR/MRV 0.246 1.896 246.2
  • PCR-amplify promoters
    • 2x 50 μL rxns each
  1. pAubR, EcoRI fwd, XbaI rev
  2. pBjaR, EcoRI fwd, XbaI rev
  3. pBraR, EcoRI fwd, XbaI rev
  4. pRpaR, EcoRI fwd, XbaI rev
Reagent Vol Mix (x2) Expected:
1. pAubR = 153
2. pBjaR = 122
3. pBraR = 214
4. pRpaR = 136
gBlock DNA (2 ng/μL) 0.5 1.0
10 μM XbaI rev 1.0 2.0
10 μM EcoRI fwd 1.0 2.0
2x GoTaq green 25.0 50.0
dH2O 22.5 8.7
  50.0 μL

Thermal cycler: Labnet - GOTAQ

  • 95°C 3 min.
  • 30x[95°C, 30 sec; 57°C 30 sec; 72°C 30 sec]
  • 72°C 3 min.
  • 4°C, ∞


  • PCR clean-up
    • Qiagen kit
    • Elute w/ 30 μL elution sln.
  • Measure conc.'s
    • Values from last week, except BraR/MRV, which was measured today
Sample OD260 260/280 ng/μL
1. pAubR PCR 0.258 1.906 257.7
2. pBjaR PCR 0.177 1.904 177.3
3. pBraR PCR 0.465 1.913 465.4
4. pRpaR PCR 0.246 1.896 246.2


  • Digests (Fermentas FD)
    • E/X
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
phosphatase 1.0
dH2O ---
  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
  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. pRpa/RpaR/MRV: pRpa(E/Xdp)/136 + RpaR/MRV(BbsI)/3980
  • Note: BraR cloning was not successful, omit from this round of assembly
  1 2 3
Insert DNA 5.0 5.0 5.0
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 (Roche) 1.0 1.0 1.0
dH2O 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, ∞





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