Team:NYMU-Taipei/Project/Phosphate/Experiments/GEL Electrophoresis

From 2008.igem.org

Contents

Experiments to be verified by Electrophoresis

  • Gene cloning (PCR)
  • Digestion for vectors and inserts (Enzyme digestion)
  • Ligation
  • Transformation (Colony PCR)

PCR for new parts

  • PST operon
    • PST left
    • PST right
  • PPK (ppk mutant = PPK 1 + PPK 2)
    • PPK 1: 09/06 ready but lost; 09/11 OK
    • PPK 2: 09/17 ready

PCR for assembly

  • Basic Parts
    • B0032 (RBS, done)
    • R0010 (pLac promoter, done)
    • R0040 (pTet promoter, done)
    • P0440 (TetR with RBS and D-term, done)
  • Composition Parts
    • existed combination
      • pLac + E0240 (from Tina)
      • pPhoB + E0240 (non-standard cutting sites: E-part-S-P from dodo)
    • new combination
    • pLac + P0440: ligation A (8+10+20 failed) and A'(10 failed)
    • pTet + E0240: ligation B (10 successful)
    • RBS + PPK: ligation C (10+20 failed)
    • RBS + PST: ligation D

PST Operon

Date and Time Gel check Protocol Comments & Actions
  • 9/1, 08 PCR
  • 9/2, 08 GEL check

NYMU 20080902 pst PCR.JPG

  • lane 1: [http://www.fermentas.com/catalog/electrophoresis/generulers.htm#1kb 1kb DNA ladder (fermentas)]
  • lane 2: PCR pst tube #1
  • lane 3: PCR pst tube #2
  • lane 4: PCR pst tube #3
  • lane 5: [http://www.fermentas.com/catalog/electrophoresis/generulers.htm#1kb 1kb DNA ladder (fermentas)]
  • Ingredients (Total 50λ)
    • 50ng/λ template 1λ
    • 10uM FP 1λ
    • 10uM RP 1λ
    • 2.5mM dNTP 2.5λ
    • 10X buffer 5λ
    • PfuTubro 1λ (2.5U)
    • ddH2O 38.5λ

  • Reaction condition
    • 95 10m, (95 30s, 55 1m, 72 3m15s) X 35, 72 5m
  • Observations
    • 3 nonspecific bands: ~500bp, 250bp and one less than 250bp (primer?)
    • Does polymerase have sufficient time to elongate the 4.6kb pst operon?

  • Actions
    • Increase extension time from 3m15s to 5m
  • 9/2, 08 PCR
  • 9/3, 08 GEL check

NYMU 20080903 pst PCR.JPG

  • lane 1: 1kb DNA ladder
  • lane 2: PCR pst tube (PfuTubro)
  • lane 3: PCR pst tube (YEA taq)
  • lane 4: 1kb DNA ladder
  • Ingredients (Total 50λ)
    • 50ng/λ template 1λ
    • 10uM FP 1λ
    • 10uM RP 1λ
    • 2.5mM dNTP 2.5λ
    • 10X buffer 5λ
    • PfuTubro 1λ (2.5U) and YEA taq
    • ddH2O 38.5λ

  • Reaction condition
    • 95 10m, (95 30s, 55 1m, 72 5m) X 30, 72 5m
  • Observations
    • taq generated a lot of non-specific DNA fragments and makes the whole lane smeared
    • the lane using pfu was not clear in running gel

  • Actions
    • Increase extension time from 5m to 10m
    • Increase pfu from 1uL to 2uL
  • 9/3, 08 PCR
  • 9/4, 08 GEL check

NYMU 20080904 pst PCR.JPG

  • lane 1: 1kb DNA ladder
  • lane 2: PCR pst (double PfuTubro)
  • Ingredients (Total 62λ)
    • 50ng/λ template 5λ
    • 10uM FP 20λ
    • 10uM RP 20λ
    • 2.5mM dNTP 5.0λ
    • 10X buffer 9.17λ
    • PfuTubro 2λ (5.0U)
    • ddH2O 0.0λ

  • Reaction condition
    • 95 10m, (95 30s, 55 1m, 72 10m) X 30, 72 5m
  • require a positive control (i.e. 3kb PCR from jesse or 2.6kb frmo tina)
  • gradient Ta (55 - 65) to find the optimal one
  • 9/5, 08 run PCR
  • 9/5, 08 GEL check

NYMU 20080905 gfp pst PCR.JPG

  • lane 1: E0240 GFP (+ control, 1114bp)
  • lane 2: pst
  • lane 3: 1Kb DNA ladder
  • Ingredients (Total 50λ)
    • 50ng/λ template 1λ
    • 10uM FP 1.0λ
    • 10uM RP 1.0λ
    • 2.5mM dNTP 2.5λ
    • 10X buffer 5.0λ
    • YEA taq 1λ (5.0U)
    • ddH2O 38.5λ

  • Reaction condition
    • 95 10m, (95 30s, 55 1m, 72 10m) X 30, 72 5m
  • 10m extension time seems not sufficient to generate 4.6kb PST
  • 9/5 GEL check

NYMU 20080905 pst re-examine.JPG

  • lane 1: 100bp DNA ladder
  • lane 2: pst by taq, 10m extension time (9/5 pst PCR)
  • lane 3: pst by double TubroPfu, 10m extension time (9/3 pst PCR)
  • two bands might be primers or short DNA fragments (~100bp)
  • adjust reaction condition
    • extension time 10m → 15m?
    • annealing temperature 50 ~ 60
  • 9/5, 9/6 PCR
  • 9/6 GEL check

NYMU 20080906 pst gradient ppk1 ppk2 htlb.jpg

  • lane 01: 1kb DNA ladder

  • lane 02: pst (Ta = 50.0°C, Textension = 15m)
  • lane 03: pst (Ta = 50.2°C, Textension = 15m)
  • lane 04: pst (Ta = 50.9°C, Textension = 15m)
  • lane 05: pst (Ta = 52.0°C, Textension = 15m)
  • lane 06: pst (Ta = 53.2°C, Textension = 15m)
  • lane 07: pst (Ta = 54.4°C, Textension = 15m)
  • lane 08: pst (Ta = 55.6°C, Textension = 15m)
  • lane 09: pst (Ta = 56.8°C, Textension = 15m)
  • lane 10: pst (Ta = 58.0°C, Textension = 15m)
  • lane 11: pst (Ta = 59.1°C, Textension = 15m)
  • lane 12: pst (Ta = 59.8°C, Textension = 15m)

  • lane 13: ppk1 (Ta = 55°C, Textension = 6m)
  • lane 14: ppk2 (Ta = 55°C, Textension = 6m)
  • lane 15: htlB (Ta = 55°C, Textension = 6m)

  • lane 16: Dye only (empty)

  • lane 17: 100bp DNA ladder

PST


  • Ingredients (Total 50λ)
    • 50ng/λ template 1λ
    • 10uM FP 1.0λ
    • 10uM RP 1.0λ
    • 2.5mM dNTP 2.5λ
    • 10X buffer 5.0λ
    • YEA taq 1λ (5.0U)
    • ddH2O 38.5λ

  • Reaction condition
    • 95 10m, (95 30s, 55 1m, 72 15m) X 30, 72 5m
  • Observations
    • pst PCR failed
      • extension time is too long so that the taq polymerases were all dead before they can amplify enough amount of DNA fragments
      • gradient search of Ta did not work
    • ppk1 PCR works
    • ppk2 PCR failed
    • htlB PCR works (positive control)

  • Actions
  • 9/8 PCR
  • 9/8 GEL check

NYMU 20080908 pst gradient and htlB.jpg

  • lane 1: 1kb DNA ladder
  • lane 2: pst (Ta = 45.0°C, Textension = 5m)
  • lane 3: pst (Ta = 47.0°C, Textension = 5m)
  • lane 4: pst (Ta = 49.4°C, Textension = 5m)
  • lane 5: pst (Ta = 51.8°C, Textension = 5m)
  • lane 6: pst (Ta = 55.0°C, Textension = 5m)
  • lane 7: htlB (Ta = 55.0°C, Textension = 5m)
  • lane 8: 100bp DNA ladder
  • Ingredients (Total 50λ)
    • 50ng/λ template 1λ
    • 10uM FP 1λ
    • 10uM RP 1λ
    • 2.5mM dNTP 2.5λ
    • 10X buffer 5λ
    • YEA taq 1λ (5.0 Unit)
    • ddH2O 38.5λ

  • Reaction condition
    • 95 2m30s, (95 30s, 55 30s, 72 5m) X 35, 72 5m
  • Observation
    • 95 2m30s is sufficient to open genomic DNA of E.coli K12
  • 9/9, 08 PCR
  • 9/9, 08 GEL check

NYMU 20080909 pst PCR.JPG

  • lane 1: [http://www.fermentas.com/catalog/electrophoresis/generulers.htm#1kb 1kb DNA ladder (fermentas)]
  • lane 2: PCR pst
  • lane 3: PCR htlB
  • lane 4: PCR ppk2
  • lane 5: 100bp DNA ladder
  • Ingredients (Total 50λ)
    • 50ng/λ template 1λ
    • 10uM FP 2.5λ
    • 10uM RP 2.5λ
    • 2.5mM dNTP 4.0λ
    • 10X buffer 5λ
    • Promega pfu 1λ (1.5U)
    • ddH2O 34.0λ

  • Reaction condition
    • 95 2m, (95 30s, 55 30s, 72 9m30s) X 20, 72 5m
  • Observations

  • Actions
  • 9/17, 08 Digestion
  • 9/17, 08 GEL check

NYMU K12 SmaI digest 02.jpg

  • lane01: 1Kb DNA marker
  • lane02: K12 (SmaI digested)
  • lane03: K12 (SmaI digested)
  • lane04: K12 (oirginal)
  • lane05: 100bp DNA marker
  • Ingredients (Total λ)
    • Stock concerntration of K12 genomic DNA:1.77mg/λ
      • template:8λ
      • NEBbuffer4:2λ
      • BSA: 2λ
      • 10units SmaI:1.416λ (1.77*8=10*X; X=1.416)
      • Total reaction volume=20λ
        • After digestion, we extracted the fragment(~7K)contained pst operon from gel for the use of template

  • Reaction condition
  • Motivation
    • digest K12 genome DNA into smaller piece containing PST operon

  • Observations

  • Actions
  • 9/17, 08 GEL extraction

NYMU K12 SmaI digest cut gel.jpg

  • cut ~6500bp - 8000bp
  • Ingredients (Total λ)

  • Reaction condition
  • Observations

  • Actions
  • 9/17, 08 PCR
  • 9/18, 08 GEL check

NYMU 20080918 ppk pst 01.jpg

  • lane 1: [http://www.fermentas.com/catalog/electrophoresis/generulers.htm#1kb 1kb DNA ladder (fermentas)]
  • lane 2: PPK overlapped PCR
  • lane 3: PPK overlapped PCR
  • lane 4: PST (template: SmaI digested K12 genomic DNA)
  • lane 5: PST (template: original K12 genomic DNA)
  • lane 6: 100bp DNA ladder
  • Ingredients (Total 50λ)
    • 3.62ng/λ SmaI digested template 1λ // 50ng/λ template 1λ
    • 10uM FP 2.5λ
    • 10uM RP 2.5λ
    • 2.5mM dNTP 4.0λ
    • 10X buffer 5λ
    • 25mM MgSO4 10λ)
    • Promega turbo 1λ (1.5U)
    • ddH2O 24.0λ

  • Reaction condition
    • 95 2m, (95 30s, 51 60s, 72 9m30s) X 20, 72 5m
  • Observations

  • Actions
  • 9/18, 08 PCR
  • 9/19, 08 GEL check

NYMU 20080919 pst.jpg

  • lane 1: [http://www.fermentas.com/catalog/electrophoresis/generulers.htm#1kb 1kb DNA ladder (fermentas)]
  • lane 2: 45°C (with Mg2+)
  • lane 3: 47°C (with Mg2+)
  • lane 4: 48.2°C (with Mg2+)
  • lane 5: 50.6°C (with Mg2+)
  • lane 6: 53°C (with Mg2+)
  • lane 7: 45°C
  • lane 8: 47°C
  • lane 9: 48.2°C
  • lane 10: 50.6°C
  • lane 11: 53°C
  • lane12:100bp
  • Ingredients (Total 25λ)
    • 50ng/λ template 0.5λ
    • 10uM FP 1.25λ
    • 10uM RP 1.25λ
    • 2.5mM dNTP 2λ
    • 10X buffer 2.5λ
    • 25mM MgSO4 5λ)
    • Promega pfu 0.5λ (1.5U)
    • ddH2O 12.0λ

  • Reaction condition
    • 95 2m, (95 30s, 45 47 48.2 50.6 53 60s, 72 9m30s) X 20, 72 5m
  • Observations

  • Actions
  • 9/30, 08 gradient PCR
  • 10/1, 08 GEL check

NYMU 20081001 PST gradient 03.jpg

  • lane 01: YEA 1Kb DNA ladder

  • lane 02: PST right (black 1)
  • lane 03: PST right (black 2)
  • lane 04: PST right (black 3)
  • lane 05: PST right (black 4)
  • lane 06: PST right (black 5)

  • lane 07: PST left (red 1)
  • lane 08: PST left (red 2)
  • lane 09: PST left (red 3)
  • lane 10: PST left (red 4)
  • lane 11: PST left (red 5)

  • lane12: HtlB (+ control)
  • Ingredients (Total 25λ)
    • 50ng/λ template ?λ
    • 10uM FP 0.5λ
    • 10uM RP 0.5λ
    • 2.5mM dNTP 1.0λ
    • 10X buffer 2.5λ
    • YEA taq 0.125λ
    • ddH2O 20.375λ

  • Reaction condition
    • 95 2m, (95 15s, 45-55 30s, 72 3m) X 30, 72 5m
  • Observations
    • no PST right segment can be amplified
    • only one PST left segment was amplified under Ta =~ 53.0

  • Actions
  • 10/1, 08 gradient PCR
  • 10/1, 08 GEL check

NYMU 20081001 PST right.jpg

  • lane 04: PST right (Ta = 45.0)
  • lane 05: PST right (Ta = 47.0)
  • lane 06: PST right (Ta = 50.6)
  • lane 07: PST right (Ta = 53.0)
  • lane 08: PST right (Ta = 55.0)

  • lane 09: HtlB (+ control)

  • lane 10: YEA 1Kb DNA ladder
  • Ingredients (Total 25λ)
    • 50ng/λ template ?λ
    • 10uM FP 0.5λ
    • 10uM RP 0.5λ
    • 2.5mM dNTP 1.0λ
    • 10X buffer 2.5λ
    • YEA taq 0.125λ
    • ddH2O 20.375λ

  • Reaction condition
    • 95 2m, (95 15s, 45;47;50.6;53;55 30s, 72 4m) X 30, 72 5m
  • Observations
    • Two PST right segments were amplified under Ta = 45.0 and 47.0

  • Actions
  • 10/5, 08 fusion PCR
  • 10/5, 08 GEL check

NYMU 20081005 PST fusion.jpg

  • lane 02: YEA 1Kb DNA ladder

  • lane 03: PST fusion (Ta = 45.0)

  • lane 04: HtlB (+ control)

  • lane 05: YEA 1Kb DNA ladder
  • Ingredients (Total 25λ)
    • 50ng/λ template ?λ
    • 10uM FP 0.5λ
    • 10uM RP 0.5λ
    • 2.5mM dNTP 1.0λ
    • 10X buffer 2.5λ
    • YEA taq 0.125λ
    • ddH2O 20.375λ

  • Reaction condition
    • 95 2m, (95 30s, 45 60s, 72 8m) X 30, 72 5m
  • Observations
    • Combining two segments of PST operon was not successful (smeared). However, there was a bright region near the expected length (4.5Kb).

  • Actions


PPK

Date and Time Gel check Protocol Comments & Actions
  • 9/2, 08 PCR
  • 9/3, 08 GEL check

NYMU 20080903 ppk pst PCR.JPG

  • lane 1: 100bp DNA ladder
  • lane 2: PCR ppk 1 (left segment, ~ 270bp)
  • lane 3: PCR ppk 2 (right segment, ~ 1797bp)
  • lane 4: PCR pst (YEA taq)
  • lane 5: PCR pst (PfuTubro)
  • lane 6: 1kb DNA ladder
  • ppk left segment(~ 270bp)
    • 95 10m, (95 30s, 55 60s, 72 1m) x 35, 72 5m
    • Promega pfu
  • ppk right segment (~ 1797bp)
    • 95 10m, (95 30s, 55 60s, 72 2m) x 35, 72 5m
    • Promega pfu
  • pst (~ 4.6kb)
    • extension time 5 mins
  • Observations
    • PCR ppk1 has a clear band at ~300bp
    • PCR ppk2 does not have any band on this lane
    • PCR pst by taq clearly has a smear lane (a lot of non-specific annealing)
    • PCR pst by TubroPfu has

  • Actions
    • increse extension time of ppk 2 (right segment)

  • Notes
    • ppk 1 (left segment) was lost >_<
  • 9/5, 08 PCR
  • 9/5, 08 GEL check

NYMU 20080905 ppk PCR.JPG

  • ppk right segment (~ 1797bp)
    • 95 10m, (95 30s, 55 60s, 72 3.5m) x 30, 72 5m
    • Promega pfu
  • lacking of positive control
  • longer extension time(~4min)
  • 9/9, 08 PCR
  • 9/9, 08 GEL check

NYMU 20080909 htlB ppk2 taq pfu 13cycle.JPG

  • lane 01: [http://www.fermentas.com/catalog/electrophoresis/generulers.htm#1kb 1kb DNA ladder (fermentas)]

  • lane 02: htlB (taq)
  • lane 03: htlB (pfu)

  • lane 04: PPK2 (taq)
  • lane 05: PPK2 (pfu)

  • lane 06: 100bp DNA ladder
  • Ingradients(Total 50λ)
    • 50ng/λ template 1λ
    • 10uM FP 2.5λ
    • 10uM RP 2.5λ
    • 2.5mM dNTP 4.0λ
    • 10X buffer 5λ
    • Promega pfu 1λ (1.5U)
    • ddH2O 34.0λ

  • Reaction condition
    • 95 1'30m, (95 30s, 51 60s, 72 5m) X 20, 72 5m
    • Suddently stop at the 13th cycle
  • Observation
    • though the PCR machine suddenly stop at the 13th cycle (Sensor Error), the positive control HtlB with Taq polymerase still have a band at 2Kb.

  • Action
    • We moved the PCR product tube to the other PCR machine, continuing the remained 7 cycles.
  • 9/9, 9/10 08 PCR
  • 9/10, 08 GEL check

NYMU 20080910 ppk2 htlB.JPG

  • lane 01: [http://www.fermentas.com/catalog/electrophoresis/generulers.htm#1kb 1kb DNA ladder (fermentas)]

  • lane 2: 9/10, ppk2 (taq)
  • lane 3: 9/10, ppk2 (pfu)
  • lane 4: 9/10, htlB (taq)
  • lane 5: 9/10, htlB (pfu)

  • lane 6: 9/9, ppk2 (taq)
  • lane 7: 9/9, ppk2 (pfu)
  • lane 8: 9/9, htlB (taq)
  • lane 9: 9/9, htlB (pfu)

  • lane 10: 100bp DNA ladder
  • Ingredients (Total 50λ)
    • 50ng/λ template 1λ
    • 10uM FP 2.5λ
    • 10uM RP 2.5λ
    • 2.5mM dNTP 4.0λ
    • 10X buffer 5λ
    • Promega pfu 1λ (1.5U)
    • ddH2O 34.0λ

  • Ingredients (Total 50λ)
    • 50ng/λ template 1λ
    • 10uM FP 1.0λ
    • 10uM RP 1.0λ
    • 2.5mM dNTP 2.5λ
    • 10X buffer 5λ
    • YEA taq 1λ (5.0U)
    • ddH2O 38.5λ

  • Reaction condition (9/9, 08)
    • 95 2m, (95 30s, 51 60s, 72 5m) X 20, 72 5m

  • Reaction condition (9/10, 08)
    • 95 2m, (95 30s, 50 60s, 72 9m30s) X 20, 72 5m
  • Observations
    • PPK2 has ~1.8kb band by taq (Ta = 51, te = 5m)
    • assume annealing problem is universal, we use taq to find a optimal Ta
    • in case we find it, we can apply this Ta to pfu PCR

  • Actions
    • use Ta gradient to find the optimal Ta
  • 9/10, 08 PCR
  • 9/10, 08 GEL check

NYMU 20080910 ppk2 gra.JPG

  • lane 01: [http://www.fermentas.com/catalog/electrophoresis/generulers.htm#1kb 1kb DNA ladder (fermentas)]

PPK2

  • lane 02: 50.9
  • lane 03: 53.2
  • lane 04: 55.6
  • lane 05: 56.8
  • lane 06: 59.1
  • lane 07: 60.0

  • lane 08: 55.6(htlB)

  • lane 09: 55.6(E0240 GFP)

  • lane 10: 100bp DNA ladder
  • Ingredients (Total 50λ)
    • 50ng/λ template 1λ (1ng/λ = 1ug/mL)
    • 10uM FP 2.5λ (0.5uM)
    • 10uM RP 2.5λ (0.5uM)
    • 2.5mM dNTP 4.0λ (200uM)
    • 10X buffer 5λ
    • NEB taq 1λ (5.0U)
    • ddH2O 34.0λ

  • Reaction condition
    • 95 2m, (95 30s, [51-59:2] 60s, 72 2m) X 20, 72 5m
  • Observations

  • Actions
    • longer extension time (2m to 5m)
    • increase the amount of cycles (20 to 25)
  • 9/10, 08 PCR
  • 9/11, 08 GEL check

NYMU 20080910 ppk2 graII.JPG

  • lane 01: [http://www.fermentas.com/catalog/electrophoresis/generulers.htm#1kb 1kb DNA ladder (fermentas)]

PPK2

  • lane 02: 47.6
  • lane 03: 51.4
  • lane 04: 52.8
  • lane 05: 54.0
  • lane 06: 56.0

  • lane 07: 54.0(htlB)

  • lane 08: 54.0(E0240 GFP)

  • lane 09: 100bp DNA ladder
  • Ingredients (Total 50λ)
    • 50ng/λ template 1λ (1ng/λ = 1ug/mL)
    • 10uM FP 2.5λ (0.5uM)
    • 10uM RP 2.5λ (0.5uM)
    • 2.5mM dNTP 4.0λ (200uM)
    • 10X buffer 5λ
    • NEB taq 1λ (5.0U)
    • ddH2O 34.0λ

  • Reaction condition
    • 95 1m, (95 30s, [47.6-56:2] 60s, 72 5m) X 25, 72 5m
  • Observations
    • we got the correct band (~1.8K) at Ta=52.8,51.4 and 47.6 degree celcius but also find that the PPK2 lanes smear serously.

  • Actions
    • substituting Taq ploymerase to Promega (pfu)
    • longer the extension time (5m to 6m30s)
  • 9/11, 08 PCR
  • 9/11, 08 GEL check

NYMU 20080910 ppk1 and ppk2.JPG

  • lane 01: 100bp DNA ladder

  • lane 02: PPK1
  • lane 03: PPK2

  • lane 04: htlB

  • lane 05: 100bp DNA ladder
  • Ingredients (Total 50λ)
    • 50ng/λ template 1λ (1ng/λ = 1ug/mL)
    • 10uM FP 2.5λ (0.5uM)
    • 10uM RP 2.5λ (0.5uM)
    • 2.5mM dNTP 4.0λ (200uM)
    • 10X buffer 5λ
    • Promega pfu 1λ (5.0U)
    • ddH2O 34.0λ

  • Reaction condition
    • 95 2m, (95 30s, 53 60s, 72 6'30m) X 25, 72 5m
  • Observations
    • there were no band on the gel, PCR condition failure.

  • Actions
  • 9/11, 08 PCR
  • 9/14, 08 GEL check

NYMU 20080911 ppk1.JPG

  • lane 01: 100bp DNA ladder

  • lane 02: htlB

  • lane 03: PPK1
  • Ingredients (Total 50λ)
    • 50ng/λ template 1λ (1ng/λ = 1ug/mL)
    • 10uM FP 2.5λ (0.5uM)
    • 10uM RP 2.5λ (0.5uM)
    • 2.5mM dNTP 4.0λ (200uM)
    • 10X buffer 5λ
    • Promega pfu 1λ (5.0U)
    • ddH2O 34.0λ

  • Reaction condition
    • 95 2m, (95 30s, 55 60s, 72 1m) X 20, 72 5m
  • Observations
    • Find the correct and clear PPK1 band at ~273bp

  • Actions
    • Store the correct PCR Product in the 1.2ml eppendorf at -20 degree celcius refrigerator
  • 9/15, 08 PCR
  • 9/15, 08 GEL check

NYMU 20080915 ppk2 htlB.JPG

  • lane 01: 100bp DNA ladder

  • lane 02: PPK2

  • lane 03: htlB

  • lane 04: 100bp DNA ladder
  • Ingredients (Total 50λ)
    • 50ng/λ template 1λ (1ng/λ = 1ug/mL)
    • 10uM FP 2.5λ (0.5uM)
    • 10uM RP 2.5λ (0.5uM)
    • 2.5mM dNTP 4.0λ (200uM)
    • 10X buffer 5λ
    • Promega pfu 1λ (5.0U)
    • ddH2O 34.0λ

  • Reaction condition
    • 95 2m, (95 30s, 51 60s, 72 6m30s) X 20, 72 5m
  • Observations
    • No band appears in lane 02

  • Actions
    • We had longer but incorrect band using promega pfu (Ta = 50 and extension time = 9m30s)
    • Increase extension time from 6m30s to 9m30s
  • 9/15, 08 PCR
  • 9/16, 08 GEL check

NYMU 20080915pstppk2.JPG

  • lane 01: 1Kbp DNA ladder

  • lane 02: Pst operon
  • lane 03: PPK2

  • lane 04: 100bp DNA ladder
  • Ingredients (Total 50λ)
    • 50ng/λ template 1λ (1ng/λ = 1ug/mL)
    • 10uM FP 2.5λ (0.5uM)
    • 10uM RP 2.5λ (0.5uM)
    • 2.5mM dNTP 4.0λ (200uM)
    • 10X buffer 5λ
    • pfu turbo 1λ (5.0U)
    • ddH2O 34.0λ

  • Reaction condition
    • 95 2m, (95 30s, 51 60s, 72 9m30s) X 20, 72 5m
  • Observations

  • Actions
    • We re-run the remaining PPK2 PCR product to make the band more clear (the DNA concentration got higher) in order to do gel extraction of specific band (1.7K)
    • Run nested PCR to amplify the PPK2 target DNA fragment
  • 9/16, 08 nested PCR
  • 9/17, 08 GEL check

NYMU 20080915ppk2nestedPCR.JPG

  • lane 01: 1Kbp DNA ladder

  • lane 02: PPK2 (K12 as template)
  • lane 03: PPK2 (PPK2 as template, nested PCR)

  • lane 04: 100bp DNA ladder
  • Ingredients (Total 50λ)
    • 50ng/λ template 1λ (1ng/λ = 1ug/mL)
    • 10uM FP 2.5λ (0.5uM)
    • 10uM RP 2.5λ (0.5uM)
    • 2.5mM dNTP 4.0λ (200uM)
    • 10X buffer 5λ
    • pfu turbo 1λ (5.0U)
    • ddH2O 34.0λ

  • Reaction condition
    • 95 2m, (95 30s, 51 60s, 72 9m30s) X 20, 72 5m
  • Observations

  • Actions
  • 9/20, 08 PCR
  • 9/21, 08 GEL check

NYMU 20080921 pst ppk PCR.jpg

  • lane 01: 1Kbp DNA ladder

  • lane 02: Pst (9/18 gradient-45)
  • lane 03: PPK overlapped(Mg2+) (PPK1(9/11) PPK2 III(9/20))
  • lane 04: PPK overlapped (PPK1(9/11) PPK2 III(9/20))

  • lane 06: HtlB

  • lane 07: 100bp DNA ladder
  • Ingredients (Total 50λ)
    • 50ng/λ template 2λ (PPK1(9/11)1λ PPK2 III(9/20)1λ)
    • 10uM FP 2.5λ (0.5uM)
    • 10uM RP 2.5λ (0.5uM)
    • 2.5mM dNTP 4.0λ (200uM)
    • 10X buffer 5λ
    • pfu promega 1λ (5.0U)
    • ddH2O 33.0λ

  • Reaction condition
    • 95 2m, (95 30s, 51 60s, 72 9m30s) X 20, 72 5m
  • Observations
    • PST (Ta=45) band disappeared
    • around 2kb has a band in PPK overlapped PCR

  • Actions
  • 9/23, 08 GEL check

NYMU 20080923 pst ppk E0240.jpg

  • lane 01: 1Kbp DNA ladder

  • lane 02: PST (9/22, KOD)

  • lane 03: PPK (Mg2+, EcoRI digested)
  • lane 04: PPK (EcoRI digested)
  • lane 05: PPK (Mg2+)
  • lane 06: PPK

  • lane 07: E0240 (D. XP)

  • lane 08: NEB 100bp DNA ladder (1517, 1200, 1000, ...)
  • Ingredients (Total 20λ)

  • Reaction condition
  • Observations
    • EcoRI digestion does not change the band pattern of PPK PCR product
    • If EcoRI works, these PPK should be mutants

  • Actions
  • 9/24, 08 GEL check

NYMU 20080924 ppk.JPG

  • lane 01: NEB 100bp DNA ladder (1517, 1200, 1000, ...)
  • lane 2,3,4,5: PPK (pfu overlapped)
  • lane 6,7: pst (KOD)
  • Observations
    • PPK indeed has a band around 2kb at lane 2-5 (cut it as insert)
    • PST does not have any band (still hanging >_<)


Colony PCR

Date and Time Gel check Protocol Comments & Actions
  • 9/19, 08 colony PCR
  • 9/20, 08 GEL check

NYMU 20080920 P0440 colony PCR.jpg

  • lane 01: 100bp
  • lane 02: positive control (E0240?)
  • lane 03 - 10: P0440 colony #1 - #8
  • lane 11: negative control (no template)
  • lane 12: 100bp
  • Ingradients

  • Reaction condition
    • 95 2m, (95 30s, 55 30s, 72 60s) X 25, 72 7m
    • we ran less cycles than usual (30 cycles)
  • Observations
    • no band at lane 03 - 10 (expected VRVF2 length: 1078)
    • positive control is not correct

  • Actions
    • increase the number of cycles to 30
  • 9/20, 08 colony PCR
  • 9/20, 08 GEL check

NYMU 20080920 P0440 J04430 I763004 colony PCR.jpg

  • lane 01: 100bp marker
  • lane 02 - 09: P0440 colony #1 - #8
  • lane 10: J04430-07 colony #1
  • lane 11: I763004-08 colony #1
  • lane 12: J04430-08 colony #1
  • lane 12 - 14: J04430-07 colony #2 - #4
  • Ingradients

  • Reaction condition
  • Observations
    • P0440 (expected VRVF2 length: 1078): no hit; one 500bp band is not correct
    • J04430 (expected VRVF2 length: 1321): no hit
    • I763004 (expected VRVF2 length: 1360): not hit
    • colony PCR protocol may work

  • Actions
    • pick more colony by the same protocol
  • 9/21, 08 colony PCR
  • 9/21, 08 GEL check

NYMU 20080921 colony PCR.jpg

  • lane 01: 100bp marker (forget to load >_<)
  • lane 02 - 08: pLac + E0240 colony #1 - #3, #9 - #12
  • lane 09 - 15: P0440 colony #9 - #15
  • lane 16 - 22: J04430-07 colony #5 - #11
  • lane 23: positive control (pPhoB + E0240 colony #2; around 1.6Kb)
  • lane 24: negative control (pUC19)
  • lane 25: 1kb marker (forget to load >_<)
  • Ingradients

  • Reaction condition
  • Observations
    • no DNA marker loaded; use positive control (1.6Kb) as marker instead
    • pLac + E0240 (expected VRVF2 length: 1320): hit ratio 7/7
    • P0040 (expected VRVF2 length: 1078): hit ratio 7/7
    • J04430 (expected VRVF2 length: 1321): hit ratio 5/7

  • Actions
  • 9/26, 08 colony PCR
  • 9/26, 08 GEL check

NYMU 20080926 colony PCR.jpg

  • lane 01: empty
  • lane 02: 1kb marker
  • lane 03 - 10: pLac + P0440 colony #1 - #8
    • 1284 bp = 200 + 6 + 840 + 238
  • lane 11 - 19: pTet + E0240 colony #1 - #8
    • 1174 bp = 54 + 6 + 876 + 238, #7 repeats twice
  • lane 20: positive control (P0440)
    • 1078 bp = 840 + 238
  • lane 21: negative control (w/o template)
  • lane 22: 100kb maker
  • lane 23: empty
  • lane 24: empty
  • lane 25: empty
  • Ingradients (0.5X)
    • VR, VF2 1 uL
    • 10X buffer 2.5 uL
    • dNTP 1 uL
    • taq 0.125 uL
    • ddH2O 20.375 uL

  • Reaction condition
    • AB new PCR machine, protocol name: colony
  • Observations
    • pLac + P0440 (expected VRVF2 length: 1284): no hit
    • pTet + E0240 (expected VRVF2 length: 1174): hit ratio 1/8
      • colony #2 of pTet + E0240 has correct length

  • Actions
    • liquid culture colony #2 of pTet + E0240 and extract plasmid
  • 9/27, 08 colony PCR
  • 9/27, 08 GEL check

NYMU 20080927 colony PCR pLac P0440 and RBS PPK.jpg

  • lane 01: 100bp marker
  • lane 02 - 11: pLac + P0440 (first ligation) colony #9 - #18
  • lane 12 - 21: RBS + PPK colony #1 - #10
  • lane 22: P0440 (positive control)
  • lane 23: negative control (w/o template)
  • lane 24: 100bp marker
  • lane 25: empty
  • Ingradients

  • Reaction condition
  • Observations
    • new ligation of pLac + P0440: no hit

  • Actions
    • re-ligate pLac + P0440
  • 9/27, 08 colony PCR
  • 9/27, 08 GEL check

NYMU 20080927 colony PCR pLac P0440.jpg

  • lane 01: 100bp marker
  • lane 02 - 11: pLac + P0440 (second ligation) colony #1 - #10
  • lane 12: P0440 (positive control)
  • Ingradients

  • Reaction condition
  • Observations

  • Actions
  • 10/1, 08 colony PCR
  • 10/1, 08 GEL check

NYMU 20081001 colony PCR pLac P0440.jpg

  • lane 01: empty
  • lane 02: 1kb marker
  • lane 03 - 22: pLac + P0440 colony #19 - #38
  • lane 23: 100kb maker
  • lane 24: empty
  • lane 25: empty
  • Ingradients (0.5X)
    • VR, VF2 1 uL
    • 10X buffer 2.5 uL
    • dNTP 1 uL
    • taq 0.125 uL
    • ddH2O 20.375 uL

  • Reaction condition
  • Observations
    • pLac + P0440 (expected VRVF2 length: 1284): no hit

  • Actions
    • re-ligation of pLac + P0440
  • 10/1, 08 colony PCR
  • 10/1, 08 GEL check

NYMU 20081001 colony PCR RBS PPK.jpg

  • lane 01: empty
  • lane 02: 1kb marker
  • lane 03 - 22: RBS + PPK colony #11 - #30
  • lane 23: 100kb maker
  • lane 24: empty
  • lane 25: empty
  • Ingradients (0.5X)
    • VR, VF2 1 uL
    • 10X buffer 2.5 uL
    • dNTP 1 uL
    • taq 0.125 uL
    • ddH2O 20.375 uL

  • Reaction condition
  • Observations
    • RBS + PPK (expected VRVF2 length: 2324): no hit

  • Actions
    • extension time (1m15s) is too short to amplify the 2.3Kb RBS + PPK
    • increase the extension time to 2m20s
    • However, the clear band at around 300bp indicates the ligation failed