Team:CMUQ/BioSensor Lab

BioSensor Lab

BioSensor Lab

A1: T7 salt sensor

Introduction

The goal is to amplify T7 salt sensor, digest it along with pSB1C3 plasmid with EcoRI and PstI

Materials

  • Polymerase: Fusion Taq or Ampli tas (gold)
  • Primers: PrefixF and SuffixR
  • Nuclease free H2O
  • 5xHF PCR Buffer
  • gBlock DNA
  • dNTPs
  • PCR tubes

Procedure

Preparation of reagents

  1. Resuspend gBLOCK in 20µL nuclease free dH2O
  2. Resuspend primers to make 100µM stock then dilute to make a 10µM stock
  3. Add the following reagents to a PCR tube:
    4. PCR reagents
    Reagent Volume (µL)
    Nuclease Free water 28.5
    5xHF PCR Buffer 10
    dNTPs 1
    Primer1 2.5
    Primer2 2.5
    gBLOCK DNA 5
    Phusion Taq, ampli taq 0.5
    Total 50
  4. Run the PCR machine
  5. Digest amplified sequence with EcoRI and PstI
  6. Digest pSB1C3 vector with EcoRI and PstI
  7. If you clone into a vector that expresses a fluorescent protein, other than red since you are cloning in red, then colonies that still express that fluorescent protein will be negative clones and the red or white colonies will be the ones that you want.
  8. Run on an agarose gel, cut out the bands and purify.
  9. Send clones for sequencing

A2: PCR Purification

Introduction

Adapted from: Here

Materials

The QIAquick PCR Purification Kit (cat. nos. 28104 and 28106) can be stored at room temperature (15–25°C) for up to 12 months.

Procedure

Notes before starting:

  1. Add ethanol (96–100%) to Buffer PE before use (see bottle label for volume).
  2. All centrifugation steps are carried out at 17,900 x g (13,000 rpm) in a conventional table-top microcentrifuge at room temperature.
  3. Add 1:250 volume pH indicator I to Buffer PB. The yellow color of Buffer PB with pH indicator I indicates a pH of ≤7.5. If the purified PCR product is to be used in sensitive microarray applications, it may be beneficial to use Buffer PB without the addition of pH indicator I. Do not add pH indicator I to buffer aliquots.

Protocol

  1. Add 5 volumes Buffer PB to 1 volume of the PCR reaction and mix. If the color of the mixture is orange or violet, add 10 μl 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn yellow.
  2. Place a QIAquick column in 􀁓 a provided 2 ml collection tube or into a vacuum manifold. For details on how to set up a vacuum manifold, refer to the QIAquick Spin Handbook.
  3. To bind DNA, apply the sample to the QIAquick column and 􀁓 centrifuge for 30–60 s or 􀁺 apply vacuum to the manifold until all the samples have passed through the column. 􀁓 Discard flow-through and place the QIAquick column back in the same tube.
  4. To wash, add 0.75 ml Buffer PE to the QIAquick column 􀁓 centrifuge for 30–60 s or 􀁺 apply vacuum. 􀁓 Discard flow-through and place the QIAquick column back in the same tube.
  5. Centrifuge the QIAquick column once more in the provided 2 ml collection tube for 1 min to remove residual wash buffer.
  6. Place each QIAquick column in a clean 1.5 ml microcentrifuge tube.
  7. To elute DNA, add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water (pH 7.0– 8.5) to the center of the QIAquick membrane and centrifuge the column for 1 min. For increased DNA concentration, add 30 μl elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge.
  8. If the purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.

A3: Gel Extraction

Introduction

Adapted from: Here

Materials

The QIAEX II Gel Extraction Kit (cat. nos. 20021 and 20051) can be stored at room temperature (15–25°C) for up to 12 months.

Procedure

Notes before starting:

  1. This protocol is for cleanup of DNA fragments of 40 bp to 50 kb. The yellow color of Buffer QX1 indicates a pH ≤7.5.
  2. Add ethanol (96–100%) to Buffer PE concentrate before use (see bottle label for volume).
  3. A heating block or water bath at 50°C is required.
  4. All centrifugation steps are carried out at 17,900 x g (~13,000 rpm) in a conventional tabletop microcentrifuge at room temperature (15–25°C).
  5. For purification of DNA from polyacrylamide gels or aqueous solutions, see the handbook.

Protocol

  1. Excise the DNA band from the agarose gel with a clean, sharp scalpel. Use a 1.5 ml microfuge tube for processing up to 250 mg agarose per tube.
  2. Weigh the gel slice in a colorless tube. Add Buffer QX1 according to DNA fragment size: 6 volumes for <100 bp; 3 volumes for 100 bp – 4 kb; 3 volumes with 2 volumes of water for >4 kb. Add 6 volumes of Buffer QX1 when using >2% or Metaphor agarose gels.
  3. Resuspend QIAEX II by vortexing for 30 s. Add QIAEX II to the sample and mix: Use 10 μl QIAEXII for ≤2 μg DNA; 30 μl for 2–10 μg DNA; and an additional 30 μl for each additional 10 μg DNA.
  4. Incubate at 50°C for 10 min to solubilize the agarose and bind the DNA. Mix by vortexing* every 2 min to keep QIAEX II in suspension. Check that the color of the mixture is yellow. If the color of the mixture is orange or violet, add 10 μl 3 M sodium acetate, pH 5.0, and mix. The color should turn to yellow. The incubation should then be continued for at least 5 min.
  5. Centrifuge the sample for 30 s and carefully remove supernatant with a pipet.
  6. Wash the pellet with 500 μl Buffer QX1. Resuspend the pellet by vortexing.* Centrifuge the sample for 30 s and remove all traces of supernatant with a pipet. This wash step removes residual agarose contaminants.
  7. Wash the pellet twice with 500 μl Buffer PE. Resuspend the pellet by vortexing.* Centrifuge the sample for 30s and carefully remove all traces of supernatant with a pipet. This step removes residual salt contaminants.
  8. Air-dry the pellet for 10–15 min or until the pellet becomes white. If 30 μl QIAEX II suspension is used, air-dry the pellet for approximately 30 min. Do not vacuum dry, as overdrying, may lead to decreased elution efficiency.
  9. To elute DNA, add 20 μl of 10 mM Tris·Cl, pH 8.5, TE buffer (10 mM Tris·Cl, 1 mM EDTA, pH 8.0), or water and resuspend the pellet by vortexing.* Incubate according to the DNA fragment size: 5 min at room temperature (15–25°C) for ≤4 kb; 5 min at 50°C for 4–10 kb; or 10 min at 50°C for >10 kb.
  10. Centrifuge for 30 s, and carefully pipet the supernatant into a clean tube. The supernatant now contains the purified DNA.
  11. Optional: repeat steps 9 and 10 and combine the eluates. A second elution step will increase the yield by approximately 10–15%.

    12. For fragments larger than 10 kb, resuspend the pellet by inverting and flicking the tube. Vortexing can cause shearing of large DNA fragments.