Antibiotic Stocks

Competent Cell Preparations

Expression of IDT Constructs

IVT Protocol

Wear gloves at all times, work at a clean bench, keep all samples in closed tubes at all times to avoid contamination, and use fresh MiliQ H2O. Thaw samples on ice. Prepare in-vitro transcription mixture by pipetting the following components in this order. Vortex after each component is added.

Materials Needed:
16.35 µL MilliQ H2O
10 µL 5X TraB transcription buffer
5 µL 100 mM DTT
6 µL 25 mM NTPs
2.5 µL 100 mM GMP
1 µL 0.5 U/mL iPPase
4 µL T7-RNA polymerase
5 µL Template DNA
0.5 µL 50 U/mL RNAse inhibitor

Procedure:
Add the components in the order described above (except for the RNAse)
Incubate the reaction at 37°C
Take sample at the following time points: 0 minutes, 4 hours, and after overnight incubation
Add 0.5 uL RNAse and incubate at 37°C for 1 hour

LB Media (2L):

21 g Tryptone
10.5 g Yeast Extract
21 g NaCl

Dissolve to 2.10L with MilliQ H2O
Adjust pH to 7 with 5M NaOH
500 mL aliquots into 4 2L flasks
50 mL aliquots into 2 125 mL flasks

Autoclave
Store at 4 °C if the autoclave is not readily available
Apply autoclave tape, and include the appropriate information (Initials/iGEM, contents)

Allow the media to cool before adding antibiotics
Add the antibiotic of choice, to the specified concentration
Ampicillin: 100 µg/mL
i.e 1 mL in 1L since the stock concentration is 100 mg/mL
Note: 100 µg/mL for high copy plasmids, and 50 µg/mL for low copy plasmids
https://openwetware.org/wiki/Ampicillin
Kanamycin: 50 µg/mL
i.e 1 mL in 1L since the stock concentration is 50 mg/mL

Store at room temperature in SynBridge

This protocol was developed from the protocols of the lab of Dr. Borries Demeler.

PCR amplification protocol.

For a 50 µL reaction volume:

To a 0.2 mL PCR tube: 30.8 µL MilliQ Water, 4.0 µL 10X PFU Bufer, 0.8 µL 10mM dNTPs, 2.0 µL Forward Primer (Prefix), 2.0 µL Reverse Primer (Suffix), 0.5 µL Template DNA (Conc. 10 ng/µL), 0.5 µL PFU

Thermocycler: 30 seconds at 95 °C, then [25 seconds at 95 °C, 25 seconds at 62 °C, 1 minute at 72 °C] 30 times, then 1 minute at 72 °C, and Hold ∞ at 4 °C.

pJET Cloning

Add the following reagents sequentially: 10 µL 2X rection buffer, 1 µL DNA fragment (50 ng/µL), Up to 17 µL MilliQ water, 1 µL DNA blunting enzyme
Vortex briefly and centrifuge for 3-5 seconds
Incubate at 70 °C for 5 minutes and chill on ice
Set up the ligation reaction on ice. Add the following to the blunting reaction mixture: 1 µL pJET1.2/Blunt Cloning Vector (50 ng/µL), 1 µL T4 DNA ligase (50 ng/µL)
Vortex briefly and centrifuge for 3-5 seconds
Incubate at room temperature for 5 minutes.
Note for DNA fragments in excess of 3 kb, ligation can be prolonged to 30 mins
Transform ligation mixture into competent cells.

Plates - LB media with Agar

1.5L
1.5 L makes three sleeves of plates, so each 500 mL will produce one sleeve of plates

In a beaker:
15 g Tryptone
7.5 g Yeast Extract
15 g NaCl

Dissolve to 1.5 L with MilliQ H2O
Adjust pH to 7 with 5M NaOH
Transfer 500 mL aliquots into 3 1L Flasks
Flasks no more than ½ full for autoclaving, just to be on the safe side

Add 7.5 g Agar to each flask

Autoclave
Store at 4 °C if the autoclave is not readily available
Apply autoclave tape, and include the appropriate information (Initials/iGEM, contents)

Allow the media to cool before adding antibiotics
Add the antibiotic of choice, to the specified concentration
Ampicillin: 100 µg/mL, i.e 1 mL in 1L since the stock concentration is 100 mg/mL
Kanamycin 50 µg/mL, i.e 1 mL in 1L since the stock concentration is 50 mg/mL
More information: https://www.addgene.org/protocols/pouring-lb-agar-plates/

Promptly pour plates into sterile Petri dishes in a fume hood

Label the plate sleeve with the antibiotic, its concentration, the date, and who made them.
Store in Synbridge/Quad fridge at 4 °C, and use within two months

This protocol was developed from the protocols of the lab of Dr. Borries Demeler.

500 mL
In a beaker:
5 g Tryptone
2.5 g Yeast Extract
5 g NaCl
7.5 g Agar

Dissolve to 500 mL with MilliQ H2O
Adjust pH to 7 with 5M NaOH
Transfer 500 mL aliquot into flask
Flasks no more than ½ full for autoclaving, just to be on the safe side

Autoclave
Store at 4 °C if the autoclave is not readily available
Apply autoclave tape, and include the appropriate information (Initials/iGEM, contents)

Q5 PCR Protocol

For a 50 µL reaction volume:

To a 0.2 mL PCR tube: 21.7 µL MilliQ Water, 10 µL 5X Enhancer, 10 µL 5X Q5 Buffer, 2.4 µL Forward Primer (Prefix), 2.4 µL Reverse Primer (Suffix), 2.0 µL dNTPs, 1.0 µL Template DNA, 0.5 µL Q5 DNA Polymerase
Thermocycler: 30 seconds at 95 °C, then [25 seconds at 95 °C, 25 seconds at 62 °C, 1 minute at 72 °C] 30 times, then 1 minute at 72 °C, and Hold ∞ at 4 °C

For a 100 µL reaction volume:

To a 0.2 mL PCR tube: 43.4 µL MilliQ Water, 20 µL 5X Enhancer, 20 µL 5X Q5 Buffer, 4.8 µL Forward Primer (Prefix), 4.8 µL Reverse Primer (Suffix), 4.0 µL dNTPs , 2.0 µL Template DNA, 1.0 µL Q5 DNA Polymerase
Thermocycler: 30 seconds at 95 °C, then [25 seconds at 95 °C, 25 seconds at 62 °C, 1 minute at 72 °C] 30 times, then 1 minute at 72 °C, Hold ∞ at 4 °C

Restriction Digest, Dephosphorylation, and Clean-up

For a 50 µL reaction:

Assemble in a MCT tube in the following order: x µL plasmid DNA (usually 1 µL), 5 µL NEB CutSmart buffer, 1 µL EcoRI, 1 µL PstI, x µL MilliQ water (final volume 50 µL).
Incubate at 37 °C for one hour.
Heat kill enzyme at 80 °C for 10 minutes.
Add 1 µL Shrimp Alkaline Phosphatase to plasmid backbone.
Incubate at 37 °C for one hour.
Heat kill enzyme at 80 °C for 10 minutes.
Follow BioBasic enzymatic clean-up protocol.

SDS PAGE Protocol

Materials Needed: 8 ml of 15% Separating Gel: 5 ml of 6% Stacking Gel: 2.8 ml ddH2O 2.9 ml ddHO, 3 ml 40% Acrylamide 0.75 ml 40% Acrylamide 2 ml 1.5 M Tris pH 8.8 1.25 ml 0.5 M Tris pH 6.8 80 µl 10% SDS 50 µl 10% SDS, 80 µl 10% APS 50 µl 10% APS, 8 µl TEMED 5 µl TEMED

Making the separating gel:
Begin with 2.8 ml of ddH2O
Add 3 ml of 40 % acrylamide/bis-acrylamide solution
Add 2 ml of 1.5 M Tris pH 8.8 and mix
Mix in 80 µl of 10% SDS †,‡
When ready to use, add 8 µl of TEMED and mix
Immediately before pouring the gel, add 80 µl of 10% APS and mix
Making the stacking gel:
Begin with 2.9 ml of ddH2O
Add 0.75 ml of 40 % acrylamide/bis-acrylamide solution
Add 1.25 ml of 0.5 M Tris pH 6.8 and mix
Mix in 50 µl of 10% SDS †,‡
When ready to use, add 5 µl of TEMED and mix
Immediately before pouring the gel, add 50 µl of 10% APS and mix ¶
The solution can be used immediately, or it can be stored for 1 week at 4 °C A small amount of bromophenol blue can be added to aid in observing the dye front while the gel is running
The solution should be poured right away as it will begin to polymerize after the addition of APS

Remove SDS-PAGE gel from glass and rinse once in ddH2O in a suitable container with a lid. Try not to use a container much larger or much smaller then the gel.
Add enough Coomassie Stain to cover the gel by 1/2 inch (~ 1.5 cm).
Microwave on high power for 40 seconds to 1 minute (until the Coomassie Stain boils).

Incubate the gel in the Coomassie stain for 5 to 10 minutes on a rocking table. If you did not microwave the Coomassie/gel, incubate for at least 1 hour.
Pour off the Coomassie Stain. The Coomassie Stain can be recycled a couple of times by filtering it.
Rinse twice in ddH2O or used Destain solution to remove Coomassie Stain from the container.
Add fresh Destain solution to cover the gel by 3/4 inch (~ 2 cm).
Tie Kimwipes in a simple knot and place 4 of them in the Destain solution around the gel. Try to avoid laying the Kimwipes on the gel as this will cause an uneven destaining.
Microwave on high power for 40 seconds to 1 minute (until the Destain boils).
Incubate the gel in the Destain solution for 10 minutes on a rocking table. If you did not microwave the Coomassie/gel, incubate for at least 1 hour.
Discard the stained Kimwipes and replace with fresh knotted Kimwipes.
Incubate a second time for 10 minutes to overnight on a rocking table. Stop whenever the level of destaining is sufficient for you. Microwave again to speed up the process.
The used Destain solution can be recycled a couple of times by storing it in a sealed container with sponges or Kimwipes to remove all traces of Coomassie Stain.

SOC Media

SOC Media - 1 L

20 g Tryptone
5 g Yeast Extract
0.5 g NaCl
0.186 g KCl
Adjust pH to 7.0
Add MilliQ water to a volume of 1 L
Autoclave

0.95 g MgCl2
3.6 g glucose
Add after autoclaving

This protocol was developed from: http://cshprotocols.cshlp.org/content/2018/3/pdb.rec098863.full?rss=1
https://www.qiagen.com/ca/resources/faq?id=d3be05d0-ec02-4ced-aabe-dcd2a1061920&lang=en

Transformation Protocol

Note: Set the water bath to 42°C and use ice slurry for more efficient heat transfer.

1. Thaw competent cells.
2. Add 1 μL of DNA to 25 μL of competent cells.
3. Incubate on ice for 30 minutes.
4. Incubate at 42°C for 45-60 seconds.
5. Incubate on ice for 5 minutes.
6. Add 400 μL of LB/SOC media. Warm media
7. Incubate while shaking at 37°C for 1 hour.
8. Pour 100 μL - 200 μL culture on agar plates, spread plate, and leave right side up for 30 minutes in the incubator.
9. After 30 minutes, put the plate upside down in an incubator at 37°C overnight.

This protocol is from the High School iGEM protocol

Dual Transformation - General Protocol-edited by Seanna Goeseels and Marcel Koupantsis

1. Thaw competent cells from -80°C freezer, and DNA samples from -20°C freezer for approximately 30 min on ice.
2. Add the calculated amount of each sample of DNA to one 50 μL aliquot of competent cells.
3. Incubate in ice-water slurry for 30 minutes.
- Set the water bath to 42°C during this time.
4. Incubate at 42°C for 45-60 seconds.
5. Incubate in ice-water slurry for 5 minutes.
6. Add 950 μL of LB media.
- SOC medium is ideal, if available
7. Incubate in a shaking incubator at 37°C, 250 rpm for 1.5 hours.
8. Pour ALL culture on agar plates, spread plate and leave right side up for 30 minutes in the incubator.
9. After 30 minutes, put the plate upside down in an incubator at 37°C overnight

Dual Transformation - 8/7/21

1. Thaw competent cells from -80°C freezer, SPCas9C1 and P22C2-1 from -20°C freezer
- Thaw for approximately 30 min.
2. Add the calculated amount of each sample of DNA to 50 μL of competent cells.
0.28 μL of P22C2-1 and 0.35 μL SPCas9C1, in this case.
3. Incubate in ice-water slurry for 30 minutes.
- Set water bath to 42°C during this time.
4. Incubate at 42°C for 45-60 seconds.
5. Incubate in ice-water slurry for 5 minutes.
6. Add 950 μL of LB media.
- SOC medium is ideal if available
7.Incubate in a shaking incubator at 37°C and 250 rpm for 1.5 hours.
8.Pour ALL culture on agar plates, spread plate and leave right side up for 30 minutes in the incubator.
9.After 30 minutes, put the plate upside down in an incubator at 37°C overnight.

Urea PAGE Protocol

Materials Needed: 4.32 g Urea, 3.375 mL 40% Acrylamide (19:1)
1.8 mL 1X TBE
Up to 9 mL MilliQ H2O
37.5 µL 10% APS
7.5 µL TEMED

Procedure:
Clean glass plates and spacers thoroughly
Hold plates by the edges and wear gloves
Rinse plates with H2O and ethanol, then set aside to dry
Assemble the glass plates with spaces in the gel caster
Dissolve urea in acrylamide, 1X TBE, and MilliQ H2O
Assemble the gel into the electrophoresis chamber and fill the tank with 1X TBE that was pre-warmed in the microwave
Flush out the wells once more w/ 1x TBE. let the empty gel run for 30 min at 300V to warm it
Mix 5 µL RNA samples with 1 µL 6x RNA gel-loading buffer and boil the samples for 3 min (98°C) then centrifuge
Load the mixture into the wells
Connect the electrodes to a power pack, turn on the power, and begin the electrophoresis run at 300V
Run gel until the marker dyes have migrated the desired distance, turn off power, disconnect leads and discard buffer.
Detach glass plates
Stain gels with SyberSafe for about 20 mins