BioSensor Lab

A1: Insertion of restrictions sites into 4 types of gBLOCK plasmids.

Introduction

PCR with prefixS and prefixR to insert EcoRI and pstI sites

Materials

• Plasmids are:
• WT proU osmolarity promoter gBLOCK
• WT proU RBS-mRFP-B0015
• proU-B0034-mRFP-B0015 gBLOCK
• proU osmolarity promoter
• Polymerase: Fusion taq
• Primers: PrefixF and SuffixR
• Nuclease free H2O
• 5x HF PCR buffer
• 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:

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

Run PCR

Step	Temperature	Time	No. of cycles
Initial Denaturation	98 °C	30 sec	1
Denaturation	98 °C	30 sec	30
Annealing	58 °C	15 sec	30
Extension	72 °C	1.5 min	30
Hold	4 °C	-	-

5. Digest amplified sequence with EcoRI and PstI and purify

A2: Amplifying the pSB1C3 plasmid (BBa_J04450)

Introduction

Adapted from: Here

Materials

• Competent Cells (50µl per sample) such as DH5α
• 1.5mL Microcentrifuge tubes
• SOC Media
• Petri plates w LB agar and antibiotic (LB+ChL plates)
• Floating Foam Tube Rack
• Ice
• Ice bucket
• Lab Timer
• 42°C water bath
• 37°C incubator
• Sterile spreader
• Pipettes and Tips (10µl, 20µl, 200µl recommended)
• Microcentrifuge

Procedure

Notes before starting:

1. Thaw frozen competent cells on ice until just thawed.
2. Gently mix the thawed competent cells by flicking the tube. Transfer 100µL to each of the chilled culture tubes.
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.
4. Immediately return the tubes to ice for 10 minutes.
5. Heat-shock the cells for 45-50 seconds in a water bath at exactly 42°C. Do not shake.
6. Immediately place the tubes on ice for 2 minutes.
7. Add 900 µL of cold (4°C) SOC medium to each transformation reaction. Incubate for 60 minutes at 37°C with shaking
8. After 1 hour, spread 200 µL onto petri-dish (LB+ChL plates) and incubate at 37°C overnight.

A3: Ligation

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.

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

A. Digest pSB1C3 vector with EcoRI and pstI

PCR Purification

Notes before starting:

• A. Add ethanol (96–100%) to Buffer PE before use (see bottle label for volume)
• B. All centrifugation steps are carried out at 17,900 x g (13,000 rpm) in a conventional table-top microcentrifuge at room temperature.
• C. 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.

PCR Protocol:

• A. 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.
• B. 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.
• C. To bind DNA, apply the sample to the QIAquick column and centrifuge for 30–60 seconds 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.
• D. To wash, add 0.75 ml Buffer PE to the QIAquick column centrifuge for 30–60 seconds or apply vacuum. Discard flow-through and place the QIAquick column back in the same tube.
• E. Centrifuge the QIAquick column once more in the provided 2 ml collection tube for 1 min to remove residual wash buffer.
• F. Place each QIAquick column in a clean 1.5 ml micro centrifuge tube.
• G. 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
• H. 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

Gel extraction using QIAEX II Gel Extraction Kit

Notes before starting:

• A.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
• B. Add ethanol (96–100%) to Buffer PE concentrate before use (see bottle label for volume)
• C. A heating block or water bath at 50°C is required
• D. 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)
• E. For purification of DNA from polyacrylamide gels or aqueous solutions, see the handbook

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%.

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