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Lactate Sensor

Lactate

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February 23, 2012

PCR amplification of the mglB gene (Glucose Binding Protein)

Reagents

  • 35 uL ddH20
  • 10 uL 5X phusion buffer
  • 1.5 uL 10 mM dNTP's
  • 1.0 uL BI 139 GBP forward primer
  • 1.0 uL BI 140 GBP reverse primer
  • 1.0 uL diluted template PJG 181
  • 0.5 uL Phusion Polymerase

Procedure

Our forward primer was designed to include a Pst1 restriction site. Our reverse primer included an Xho1 restriction site.

Program-Phusion

1. 98 °C, 2 min

2. 98 °C, 30 sec

3. 60 °C, 30 sec

4. 72 °C, 2 min

5. repeat (2-4) 35x

6. 72 °C, 5 min

7. 4 °C, forever

March 2, 2012

PCR purification of Glucose Binding Protein (GBP)

Reagents

  • 35 uL ddH20
  • 10 uL 5X phusion buffer
  • 1.5 uL 10 mM dNTP's
  • 1.0 uL BI 139 GBP forward primer
  • 1.0 uL BI 140 GBP reverse primer
  • 1.0 uL diluted template PJG 181
  • 0.5 uL Phusion Polymerase

Procedure

March 8, 2012

Restriction digest of GBP and PBAD

PCR Product

  • 14 μL H2O
  • 5 μL 10x NEB buffer 3
  • .5 μL 100x BSA
  • 30 μL DNA
  • Mix well before adding restriction enzymes
  • 1-2 μL of each restriction enzyme
  • incubate at 37 °C for 1.5 hours. (overnight if needed)

March 9, 2011

Low-melt gel

Ran our restriction digests of our GBP gene as well as our plasmid to get rid of cut off DNA and any other wanted items. We did have bright DNA bands on the gel, and were successful in cutting out our DNA and getting them into labeled tubes.

March 13, 2012

Ligation of GBP and pBAD plasmid

Reagents

  • 6.5 uL ddH20
  • 1.5 uL 10X ligase buffer (includes ATP)
  • 1.0 uL T4 DNA ligase
  • 3.0 uL vector
  • 3.0 uL insert

Procedure

  1. Add appropriate amount of deionized H2O to sterile 0.6 mL tube
  2. Add 1 μL ligation buffer to the tube.
    Vortex buffer before pipetting to ensure that it is well-mixed.
    Remember that the buffer contains ATP so repeated freeze, thaw cycles can degrade the ATP thereby decreasing the efficiency of ligation.
  3. Add appropriate amount of insert to the tube.
  4. Add appropriate amount of vector to the tube.
  5. Add 0.5 μL ligase.
    Vortex ligase before pipetting to ensure that it is well-mixed.
    Also, the ligase, like most enzymes, is in some percentage of glycerol which tends to stick to the sides of your tip. To ensure you add only 0.5 μL, just touch your tip to the surface of the liquid when pipetting.
  6. Let the 10 μL solution sit at 22.5°C for 30 mins
  7. Denature the ligase at 65°C for 10min
  8. Dialyze for 20 minutes if electroporating
  9. Use disks shiny side up
  10. Store at -20°C

March 15, 2012

Transformation of ligation into DH5α cells

16-4JR GBP transformation and control

Procedure
  • Thaw DH5α cells on ice
  • Place 2μL of Ligation mix into ~25μL of DH5α cells
  • Vortex, or mix with flicking, then place back on ice for 5-10 mins
  • Heat shock at 42 °C for 60s
  • Place immediately back on ice for 2-5 mins
  • Add .5mL of LB and incubate at 37 °C. (30 mins if ampicillin resistant. 60 mins everything else.)
  • Plate

March 16, 2012

Streak Colonies

17-1JR

Procedure
  • pick up colony with pipette tip
  • place in 50 μL ddH2O0
  • Streak on LB amp plate
  • place streaked plate in 30 °C incubator over weekend

Arabinose Primers

17-2JR

  • Forward: BI151
  • Reverse: BI152
  • araF gene

Colony PCR

17-3JR

  • 2 μL of colony in ddH2O to --- μL master mix
  • Taq polymerase
  • run at Taq settings

April 27, 2012

PCR Purification

4/27JR 1

  • 5:1 Buffer PB to PCR
    • 200 μL Buffer to 40 μL PCR
  • Centrifuge 30-60s at 13,000 rpm
  • discard overflow
  • add .75ml buffer PE
  • centrifuge 30-60s @13,000 rpm
  • discard and centrifuge again for 1 min
  • place in 1.5ml tube
  • add 50 μL EB buffer
  • let sit for 1 min and then centrifuge 1 min

Restriction Digest

4/27JR 2

  • Same protocol as March 8, 2012 for GBP
  • Buffer 3. Restriction sites are pstI and XhoI

Josh's Primers

  • design site directed mutagenesis primers

April 30, 2012

Low Melt Gel AraF

  • 1% low melt agarose gel
  • 6 μL Loading Dye
  • 6 μL 1 kb ladder
  • 50 μL Restriction Digest of AraF
  • Run @ 90V for 52 mins.

May 1, 2012

Ligation

  • control (w/out vector)
  • ligation of araF low melt Restriction Digest

Transformation

  • transformed plasmid into DH5α cells
  • plated 500 μL each of control and araF pBAD plasmid
  • placed in 30 °C incubator till Friday

May 8, 2012

Cell growth

  • placed colonies 1, 3, and 4 in separate 5 mL of LB/AMP.
  • Grew overnight in 37 °C incubator

May 9, 2012

Plasmid Isolation

  • used plasmid mini prep kit
  • isolated plasmid from 3 colonies

==May 11, 2012

Repeat cell growth

  • ran nano drop on 3 cell colonies
    • only had 9 ng/μL
  • began re-growth of colonies 1, 3 and 4.
  • placed in 5 mL LB/AMP and put in 37 °C incubator

May 12, 2012

Plasmid prep

  • Took LB cells and isolated plasmid
  • used plasmid mini prep kit
  • obtained around 80 ng/μL
    • error for each sample on N.D. machine. Will re-run Monday

May 14, 2012

Nano Drop re-read

  • re-took N.D. readings with success
    • 1 153 ng/μL
    • 3 96 ng/μL
    • 4 114 ng/μL

May 15, 2012

Sequence Prep

  • placed 2 μL of each isolated plasmid
  • 1 μL of forward primer for pBAD plasmid
  • labeled JR 1, 3, 4. On the side ABP

May 18, 2012

Primers

  • designed primers for trg/envz protein

May 21, 2012

  • presentation

May 22, 2012

Colony PCR

  • re-performed colony pcr with araf gene plasmids.
  • ran pcr with IG 57 and IG () primers
  • plated 8 colonies on ampacillin plate

May 29, 2012

Analysis Colony PCR

  • 1-7 incorporated araF gene
  • lane 8 did not incorporate

Growth of cells

  • chose cells 1-3 and 7 to grow for isolation of plasmid which incorporated my gene

May 30, 2012

plasmid isolation

  • performed plasmid mini prep w/kit
  • ran nano drop and obtained 140-160 ng/μL plasmid

June 4, 2012

Sequence

  • prepared 4 samples from isolated plasmids for sequencing
  • 2 μL Dna
  • 1 μL IG57 primer

June 5, 2012

Mutagenesis

  • prepared multi-mutagenesis on plasmid 1 and 2
  • ran both forward and reverse reactions for pcr

June 8, 2012

DpnI restriction

  • added 1μL of DpnI restriction enzyme to each forward and reverse pcr reaction.
  • incubate 6 hrs at 37 °C

June 11, 2012

Ligation

  • thaw DH5α cells on ice
  • add 2μL of DpnI restriction pcr reaction to cells
  • vortex or flick and sit on ice 5-10 mins
  • heat shock 42°C for 60 seconds
  • place immediately on ice 2-5 mins
  • add .5mL of LB
  • incubate 37°C for 30 mins
  • plate

June 18, 2012

Incubation

  • Had 3 colonies on ABP mutation Reverse #1
  • incubate colony in 5 mL overnight

June 19, 2012

Plasmid prep and sequence

  • used plasmid kit to isolate plasmids with mutations
  • placed 2 μL of DNA w/ 1 μL primers IG57 and IG12