Difference between revisions of "Team:CMUQ/protocols"

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. 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:

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.

A2: PCR Purification

Introduction

Adapted from:

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.