BioSensor Lab

Introduction

The main goal is to amplify and insert EcoRI and PstI restriction sites in order to enable ligation on to pSB1C3 plasmid

Preparations

LB+ ChL plates preparation

Materials

Antibiotic: Chloramphenicol powder
70% ethanol
De-ionized water
LB-Agar2
Plates with lids
Erlenmeyer flasks
60oC water bath
Autoclave tape
Autoclave
Flame

Procedure

Preparation of 250 mL LB-Agar:

Add the following reagents into a 500 mL Erlenmeyer flask:

Reagent	Mass (g) or Volume (mL)
Tryptone	2.50 g
Yeast Extract	1.25 g
Sodium Chloride (NaCl)	2.50 g
Agar	3.75 g
Thymine (2mg/mL)	1.75 mL
1N NaOH	0.50 mL
De-Ionized Water	250 mL

Swirl the Erlenmeyer flask and cover the opening with aluminum foal and tape the bottle with autoclave tape, labelled with the initials, the date and the bottle contents
Place the gel mixture in the autoclave and run on a setting that gets the sample to at least 121oC under 20 psi for at least 30 minutes. High pressure prevent the gel mixture from boiling over at high temperature.
Turn ON the water bath and heat to 60oC
After 1- 1:30 hours, the LB agar autoclave is complete, pick the Erlenmeyer flask using rubber hot hand protector and place it on the water bath

Preparation of Chloramphenicol antibiotic:

Weigh out 12.5 mg of chloramphenicol powder placed at 4oC fridge into a 2.5 mL micro centrifuge tube.
Dissolve in 500 µL 70% ethanol to make 25 mg/mL.
Vortex to mix and leave on a rack for 10 minutes while the pouring station is prepared.

Pour Plating:

Spray down the bench with bleach and 70% ethanol solution and wipe down with a paper towel to avoid contamination
Obtain a 20 small stack plates, and stack them in the hood.
Open the plates and place the lids on the side.
Obtain LB Agar flask from 60oC water bath and add 250 µL of 25 mg/mL Chloramphenicol, swill to mix.
Pour directly from the Erlenmeyer flask until a little more than half of the plate is covered.
Once all the plates are poured, ignite the flame and quickly run over plates to avoid contamination.
Leave the plates for 20 minutes on the hood to solidify at room temperature.
Cloth the plates with the lids, and stack on top of each other.
Label the plates with blue and orange markers coding for Chloramphenicol LB, the date and the initials.
Place the on a separate box and store in 4oC fridge

Making competent DH5α cells

Preparation of Agarose gel

Materials

Agarose
De-ionized water
Ethidium Bromide
10 x Tris-Borate EDTA (TBE)
Gel box and power
Gel apparatus containing box and power

Procedure

Weigh appropriate mass of Agarose into an Erlenmeyer flask
Dissolve in the appropriate volume of diH2O and 10% TBE.

For example; to make 1% mini gel: 0.5g agarose is dissolved in 45mL of dH2O and 5mL 10xTBE

Place the mixture in the microwave for 2 minutes; take out the sample mixture every 30 seconds and swirl to dissolve.
Using hot hand protector, take the sample mixture to the bench and let it cool.
Add the required volume of ethidium bromide and swirl gently to mix.
Pour immediately onto a previously setup gel apparatus by setting the comb with the box.
Allow to solidify for at least 20 minutes
Prepare 1% TBE buffer necessary for running the gel by mixing it with dH2O.

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:

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

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	-	-

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

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

1. Run the digested pSB1C3 vector with EcoRI and pstI on 1% agarose gel using 1 Kb N3232S ladder.

2. Cut of the band and purify. The band size was 2029 bp fragment

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

3. From the gel, extract the 2029 bp fragment for further analysis

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

4. Attach the extracted DNA to the gBLOCK by sending them to sequencing

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)

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

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.

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

Competent cells should have an efficiency of 1.5x108 to 6x108 cfu/µg DNA, where "cfu" means "colony-forming unit" and is a measurement of cells.

A5: Transformation into competent DH5α

Materials

Resuspended DNA to be transformed
10pg/µl Positive transformation control DNA (e.g. pSB1C3 w/ BBa_J04450, RFP on high-copy chloramphenicol resistant plasmid
Competent Cells (50µl per sample)
1.5mL Microcentrifuge tubes
SOC Media
Petri plates w/ LB agar and antibiotic
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

Resuspend DNA in selected wells in the Distribution Kit with 10µl dH20. Pipet up and down several times, let sit for a few minutes. Resuspension will be red from cresol red dye.
Label 1.5mL microcentrifuge tubes with initials and dates. Fill lab ice bucket with ice, and pre-chill 1.5 mL tubes in a floating foam tube rack.
Thaw competent cells on ice: This may take 10-15 minutes for a 260 µL stock. Dispose of unused competent cells. Do not refreeze unused thawed cells as it drastically reduces transformation efficiency.
Pipette 50 µL of competent cells into 1.5 mL tubes. Tubes should be labelled, pre-chilled in a floating tube rack for support. Keep all tubes on ice. And don’t forget the control.
Pipette 1 µL of resuspended DNA into 1.5 mL tube. Pipette from well into appropriately labeled tube. Gently pipette up and down a few times. Keep all tubes on ice.
Pipette 1 µL of control DNA into 2 mL tube. Pipette 1µl of 10pg/µl control into your control transformation. Gently pipette up and down a few times. Keep all tubes on ice
Close 1.5 mL tubes and incubate them on ice for 30 minutes. The tubes may be gently agitated/flicked to mix solution, but should be return to ice immediately
Heat shock the tubes at 42°C for 45 seconds. 1.5ml tubes should be in a floating foam tube rack. Place in water bath to ensure the bottoms of the tubes are submerged
Incubate on ice for 5 minutes and return transformation tubes to ice bucket
Pipette 100 µL of each transformation onto LB+ChL plates spread with sterilized spreader or glass beads immediately. This helps ensure that you will be able to pick out a single colony

If higher concentration needed: spin down cells at 6800g for 3mins and discard 800µL of the supernatant. Resuspend the cells in the remaining 100µL, and pipette each transformation onto petri plates Spread with sterilized spreader or glass beads immediately. This increases the chance of getting colonies from lower concentration DNA samples.

Incubate transformation plates overnight (14-18hr) at 37°C upside down. If incubated for too long, colonies may overgrow and the antibiotics may start to break down; un-transformed cells will begin to grow.

Team:CMUQ/BioSensor Lab

BioSensor Lab