BioSensor Lab
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
- Resuspend gBLOCK in 20µL nuclease free dH2O
- Resuspend primers to make 100µM stock then dilute to make a 10µM stock
- Add the following reagents to a PCR tube:
- Run the PCR machine
- Digest amplified sequence with EcoRI and PstI and purify
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 |
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 | - | - |
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
Procedures
- Thaw frozen competent cells on ice until just thawed.
- Gently mix the thawed competent cells by flicking the tube. Transfer 100µL to each of the chilled culture tubes.
- Add 1-50 ng of DNA or 1µL (0.1ng) of competent cells Control DNA per 100µL of competent cells. Quickly flick the tube several times
- Immediately return the tubes to ice for 10 minutes.
- Heat-shock the cells for 45-50 seconds in a water bath at exactly 42°C. Do not shake.
- Immediately place the tubes on ice for 2 minutes.
- Add 900 µL of cold (4°C) SOC medium to each transformation reaction. Incubate for 60 minutes at 37°C with shaking
- 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:
- 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
- Add ethanol (96–100%) to Buffer PE concentrate before use (see bottle label for volume)
- A heating block or water bath at 50°C is required
- 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)
- For purification of DNA from polyacrylamide gels or aqueous solutions, see the handbook
Gel Extraction Protocol
- A. 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.
- B. 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.
- C. 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.
- D. 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.
- E. Centrifuge the sample for 30 s and carefully remove supernatant with a pipet.
- F. 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.
- G. 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.
- H. 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.
- I. 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.
- J. Centrifuge for 30 s, and carefully pipet the supernatant into a clean tube. The supernatant now contains the purified DNA.
- K. 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
B. Ligation by using Quick ligation protocol (M2200)
- Set up the following reaction in a microcentrifuge tube on ice. (Note that the table shows a ligation using a molar ration of 1:3 vector to insert for the indicated DNA sizes)
- Gently mix the reaction by pipetting up and down and microfuge briefly
- Incubate at room temperature (25oC) for 5 minutes
- Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells. Alternatively, Store at -20oC
- Do not heat inactivate – heat inactivation dramatically reduces transformation efficiency.
Component | 20 μl REACTION |
---|---|
Quick Ligase Reaction Buffer (2X)* | 10 μl |
Vector DNA (4 kb) | 50 ng |
Insert DNA (1 kb) | WT proU osmolarity promoter = 23.04 ng proU-B0034-mRFP-B0015 = 83.40 ng proU osmolarity promoter = 83.40 ng |
Nuclease-free Water | To 20 μl |
Quick Ligase | 1 μl |
A4: Competent Cells Test
Introduction
The kit includes three vials of purified plasmid DNA from BBa_J04450 (RFP construct) in plasmid backbone pSB1C3. Each vial contains 50 µL of DNA at a different concentration: 100 pg/µL, 50 pg/µL, 10 pg/µL. Perform transformations with each of these to determine how efficient your competent cells are
Materials
- 70% ethanol
- Ice
- Ice container
- Paper towels
- Lab marker / Sharpie
- 1.5 mL microcentrifuge tube
- Competent cells aliquot(s)
- Agar plates with chloramphenicol
- SOC media
- Sterile glass beads or sterile cell spreader
- Pipettes
- Pipette tips
- 37oC Incubators (oven and shaker)
- 42oC Water-bath (or hot water source and thermometer)
Procedure
- Clean your working area by wiping down with 70% ethanol
- Thaw competent cells on ice.
- Label one 1.5 mL microcentrifuge tube for each transformation and then pre-chill by placing the tubes on ice.
- Do triplicates (3 each) of each concentration if possible, so you can calculate an average colony yield.
- Spin down the DNA tubes from the Competent Cell Test Kit/Transformation Efficiency Kit to collect all of the DNA into the bottom of each tube prior to use. A quick spin of 20-30 seconds at 8,000-10,000 rpm will be sufficient
- Note: There should be 50 µL of DNA into each microcentrifuge tube
- Pipet 1 μL of DNA into each microcentrifuge tube
- Pipet 50 μL of competent cells into each tube. Flick the tube gently with your finger to mix
- Incubate on ice for 30 minutes
- Pre-heat waterbath now to 42°C. Otherwise, hot water and an accurate thermometer works, too!
- Heat-shock the cells by placing into the waterbath for 45 seconds (no longer than 1 minutes). Be careful to keep the lids of the tubes above the water level, and keep the ice close by.
- Immediately transfer the tubes back to ice, and incubate on ice for 5 minute
- Add 950 μL of SOC media per tube and incubate at 37oC for 1 hour shaking at 200-300 rpm
- Prepare the agar plates during this time: label them, and add sterile glass beads if using beads to spread the mixture
Next day:
- Count the number of colonies on a light field or a dark background, such as a lab bench. Use the following equation to calculate your competent cell efficiency. If you've done triplicates of each sample, use the average cell colony count in the calculation.
- Efficiency (in cfu/µg) = [colonies on plate (cfu) / Amount of DNA plated (ng)] x 1000 (ng/µg)
- Note: The measurement "Amount of DNA plated" refers to how much DNA was plated onto each agar plate, not the total amount of DNA used per transformation. You can calculate this number using the following equation: Amount of DNA plated (ng) = Volume DNA added (1 µL) x concentration of DNA (refer to vial, convert to ng/µL) x [volume plated (100 µL) / total reaction volume (1000 µL)]
Results