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

• 9/1, 08 PCR
• 9/2, 08 GEL check

• 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

• 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

• 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

• 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

• 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

• lane 01: 1kb DNA ladder

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

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

• lane 1: 1kb DNA ladder
• lane 2: pst (Ta = 45.0°C, T_extension = 5m)
• lane 3: pst (Ta = 47.0°C, T_extension = 5m)
• lane 4: pst (Ta = 49.4°C, T_extension = 5m)
• lane 5: pst (Ta = 51.8°C, T_extension = 5m)
• lane 6: pst (Ta = 55.0°C, T_extension = 5m)
• lane 7: htlB (Ta = 55.0°C, T_extension = 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

• 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

• 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

• cut ~6500bp - 8000bp

• Ingredients (Total λ)

• Reaction condition

• Observations

• Actions

• 9/17, 08 PCR
• 9/18, 08 GEL check

• 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

• 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

• 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

• 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

• 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

• 9/2, 08 PCR
• 9/3, 08 GEL check

• 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

• 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

• 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

• 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

• 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

• 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

• 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

• 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

• 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

• 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

• 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

• 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

• 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

• 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 >_<)

• 9/19, 08 colony PCR
• 9/20, 08 GEL check

• 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

• 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

• 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

• 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

• 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

• 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

• 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

• 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