Difference between revisions of "Team:DTU-Denmark/Experiments"

Adapted from iGEM 3A assembly and iGEM Victoria 3A protocol.

Materials

Digestion

• Cutsmart buffer
• SpeI, XbaI, PstI and EcoRI restriction enzymes
• Biobrick compatible genes
• Milli-Q water
• 2 DNA fragments with verification primers
• PCR tubes (2 per gene fragment: 1 for digestion, 1 for ligation)

Ligation

• T4 ligase buffer
• T4 ligase

Transformation

• Competent cells
• Ice

Procedure

Digestion

1. Mix the assembly parts following their respective tables a PCR tube.
   *100 ng is needed.
   
   Upstream fragment

   Reagent	Volume
   For each gene fragment
   Milli-Q water	Up to 10 ul
   Cutsmart buffer	1 µl
   Gene	*
   SpeI	0,2 µl
   EcoRI	0,2 µl
   Total Volume	10 µl

   
   Downstream fragment

   Reagent	Volume
   For each gene fragment
   Milli-Q water	Up to 10 ul
   Cutsmart buffer	1 µl
   Gene	*
   SpeI	0,2 µl
   EcoRI	0,2 µl
   Total Volume	10 µl

   
   Backbone

   Reagent	Volume
   For every other gene fragment
   Milli-Q water	Up to 10 ul
   Buffer	1 µl
   Gene	*
   EcoRI	0,2 µl
   PstI	0,2 µl
   Total Volume	10 µl

2. Digest at 37°C for 1 hour.
3. Heat inactivate at 80°C for 20 min.
4. Store in the freezer or use for ligation.

Ligation

5. Mix the following in a PCR tube.

   Reagent	Volume
   For each of Digested DNA fragments
   Milli-Q water	up to 20 µl
   T4 DNA ligase Buffer	2.0 µl
   Backbone	2 µl
   Upstream fragment	2 µl
   Downstream fragment	2 µl
   T4 DNA ligase	1  µl
   Total Volume	20  µl

6. Incubate at 25°C for 30 min.
7. Inactivate at 65°C for 20 min

Transformation

8. 5 µl of the ligated parts is transferred to a chilled Eppendorf tube.
9. 1 µl of the positive control is transferred to an Eppendorf tube.
10. 50 µl competent cells is transferred to each tube.
    Competent cells must be held on ice!
11. Mix gently by rolling the tubes by hand.
12. Put the tubes back on ice for 30 minutes.
13. Make sure the heating block is heated up to 42°C and heat-shock for 30 seconds.
14. Place the tubes back on ice for 5 minutes.
15. Pipette 500 µl of sterile LB media to each tube.
16. Incubate for 2 hours for 37°C with shaking (this step can be shortened if an amp-backbone is used).
17. Next step is to plate it on to the plates.
18. Shake the tube with the cells gently. Pipette 100 µl cell culture on each plate.
19. Incubate at 37°C O/N.

Adapted from Qiagen's QIAprep® Spin Miniprep Kit
This protocol assumes using a centrifuge, and not vacuum manifold processing.

Materials

Buffers

• P1 buffer
• P2 buffer
• N3 buffer
• LyseBlue reagent
• PB buffer
• PE buffer
• EB buffer

Tubes

• Centrifuge tubes
• 1.5 mL Eppendorf tubes

Procedure

Quick-start protocol

1. Pellet 1–5 ml bacterial overnight culture by centrifugation at >8000 rpm (6800 x g) for 3 min at room temperature (15–25°C).
2. Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a microcentrifuge tube.
3. Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times until the solution becomes clear. Do not allow the lysis reaction to proceed for more than 5 min. If using LyseBlue reagent, the solution will turn blue.
4. Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times. If using LyseBlue reagent, the solution will turn colorless.
5. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
6. Apply 800 μl supernatant from step 5 to the QIAprep 2.0 spin column by pipetting. Centrifuge for 30–60 s and discard the flow-through.
7. Recommended: Wash the QIAprep 2.0 spin column by adding 0.5 ml Buffer PB. Centrifuge for 30–60 s and discard the flow-through.
8. Wash the QIAprep 2.0 spin column by adding 0.75 ml Buffer PE. Centrifuge for 30–60 s and discard the flow-through. Transfer the QIAprep 2.0 spin column to the collection tube.
9. Centrifuge for 1 min to remove residual wash buffer.
10. Place the QIAprep 2.0 column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μl Buffer EB (10 mM TrisCl, pH 8.5) or water to the center of the QIAprep 2.0 spin column, let stand for 1 min, and centrifuge for 1 min.
11. If the extracted 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.

Adapted from IDT's HiFi assembly protocol

Materials

Consumables

• X PCR tubes for each reaction + 1 for positive control
• X Eppendorf tube for each reaction + 1 for positive control
• X selection plate for each reaction
• 1 Amp plate for positive control

Chemicals

• Hifi DNA assembly Master mix
• Milli-Q water
• Competent E. coli (e.g. DH5α)
• Prepared DNA fragments for assembly (See information on primer construction)

Procedure

Assembly protocol

1. Set up the following reaction on ice:

   	Recommended amount of fragments used for assembly
   	2-3 Fragments*	4-6 Fragments	Positive control
   Recommended DNA Molar Ratio	Vector:insert = 1:2	Vector:insert = 1:1
   Total amount of DNA fragments	0.03-0.3pmols X uL	0.2-0.5 pmols X uL	10 uL
   NEB Hifi Assembly master mix	10 uL	10 uL	10 uL
   Milli-Q water	10-X uL	10-x uL	0 uL
   Total volume	20 uL**	20 uL**	20 uL

   * If the inserts are less than 200 bp, use a 5-fold excess of inserts instead of a 2-fold excess.
   ** If a greater number of fragments are assembled, increase the volume of the reaction and use additional HiFi DNA assembly master mix.
2. Incubate the reaction samples in a thermocycler at 50°C for 15 minutes (when 2-3 fragments are assembled) or 60 minutes (when 4-6 fragments are assembled). Following incubation, store the reaction samples at -20°C for subsequent transformation.
   Note: Extended incubation up to 60 minutes can in some cases improve transformation efficiency.

Transformation protocol

3. Thaw chemically-competent cells on ice.
4. Add 2 µL of the chilled assembly product to the competent cells. Mix gently by pipetting up or down or by flicking the tube 4-5 times. Do NOT vortex.
5. Place the mixture on ice for 30 minutes. Do not mix.
6. Heat shock at 42°C for 30 seconds. Do not mix.
7. Transfer tubes to ice for 2 minutes.
8. Add 950 µL of room temperature SOC media to the tubes.
9. Incubate the tube for 37°C for 60 minutes. shake vigorously (250 rpm) or rotate.
10. Warm selection plates to 37°C.
11. Spread 100 µL of the cells onto the selection plates.
    Note: Use Amp plates for the positive control.
12. Incubate overnight at 37°C.

Adapted from The Chen Laboratory's QIAquick Gel Extraction Kit Protocol.

Materials

Consumables

• 2*Ν 1.5 mL Eppendorf tubes
• Ν spin columns with collection tubes

Chemicals

• Buffer QG
• Buffer PE
• Buffer EB
• Isopropanol
• (potentially: 3 M sodium acetate - see procedure step 4)

Instruments

• Thermocycler/thermoshaker
• Scalpel
• Table centrifuge for 1.5 mL microcentrifuge/Eppendorf tubes.

Procedure

Gel extraction

1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel. Minimize the size of the gel slice by removing extra agarose.
2. Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg ~ 100 µl).
   For example, add 300 µl of Buffer QG to each 100 mg of gel. For >2% agarose gels, add 6 volumes of Buffer QG. The maximum amount of gel slice per QIAquick column is 400 mg; for gel slices >400 mg use more than one QIAquick column.
3. Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). To help dissolve gel, mix by vortexing the tube every 2–3 min during the incubation. IMPORTANT: Solubilize agarose completely. For >2% gels, increase incubation time.
4. After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose). If the color of the mixture is orange or violet, add 10 µl of 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn to yellow.
   Note: The adsorption of DNA to the QIAquick membrane is efficient only at pH ≤7.5. Buffer QG contains a pH indicator which is yellow at pH ≤7.5 and orange or violet at higher pH, allowing easy determination of the optimal pH for DNA binding.
5. Add 1 gel volume of isopropanol to the sample and mix.
   For example: if the agarose gel slice is 100 mg, add 100 µl isopropanol. This step increases the yield of DNA fragments <500 bp and >4 kb. For DNA fragments between 500 bp and 4 kb, the addition of isopropanol has no effect on yield. Do not centrifuge the sample at this stage.
6. Place a QIAquick spin column in a provided 2 ml collection tube.
7. To bind DNA, apply the sample to the QIAquick column, and centrifuge for 1 min at ≥10,000 x g (~13,000 rpm). The maximum volume of the column reservoir is 800 µl. For sample volumes of more than 800 µl, simply load and spin again.
8. Discard flow-through and place QIAquick column back in the same collection tube.
9. Optional: Add 0.5 ml of Buffer QG to QIAquick column and centrifuge for 1 min ≥10,000 x g (~13,000 rpm). This step will remove all traces of agarose. It is only required when the DNA will subsequently be used for direct sequencing, in vitro transcription or microinjection.
10. To wash, add 0.75 ml of Buffer PE to QIAquick column and centrifuge for 1 min ≥10,000 x g (~13,000 rpm). Note: If the DNA will be used for salt-sensitive applications, such as blunt-end ligation and direct sequencing, let the column stand 2–5 min after the addition of Buffer PE, before centrifuging.
11. Discard the flow-through and centrifuge the QIAquick column for an additional 1 min at ≥10,000 x g (~13,000 rpm). IMPORTANT: Residual ethanol from Buffer PE will not be completely removed unless the flow-through is discarded before this additional centrifugation.
12. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.
13. To elute DNA, add 50 µl of Buffer EB (10 mM Tris·Cl, pH 8.5) or H₂O to the center of the QIAquick membrane and centrifuge the column for 1 min at maximum speed. Alternatively, 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 for 1 min. IMPORTANT: Ensure that the elution buffer is dispensed directly onto the QIAquick membrane for complete elution of bound DNA. The average eluate volume is 48 µl from 50 µl elution buffer volume, and 28 µl from 30 µl. Elution efficiency is dependent on pH. The maximum elution efficiency is achieved between pH 7.0 and 8.5. When using water, make sure that the pH value is within this range, and store DNA at –20°C as DNA may degrade in the absence of a buffering agent. The purified DNA can also be eluted in TE (10 mM Tris·Cl, 1 mM EDTA, pH 8.0), but the EDTA may inhibit subsequent enzymatic reactions.

Adapted from NEB Q5 High-Fidelity 2X Master Mix

Materials

Tubes

• PCR tubes (1 per reaction)

Reagent

• Milli-Q water

DNA

• VR/VF primers
• Ligation mix

Procedure

Q5 PCR amplification with standard primers

1. Determine the needed amount (x) of template DNA via the table below. Added volumes of about 1 µl are preferred, so dilute the sample if necessary with Milli-Q water.
   
   Template amounts

   Template source	Template amount
   Genomic	1 ng - 1 μg
   Plasmid or viral	1 pg - 1 ng

2. Mix the reagents in a PCR tube.
   
   PCR reagents

   Reagent	Volume
   For each of Digested DNA fragment
   Milli-Q water	up to 50 µl
   VF primer	2.5 µl
   VR primer	2.5 µl
   Template sample	x
   Q5 2X Master Mix	25 µl
   Total Volume	50  µl

3. For PCR machines without heated lid: overlay the sample with mineral oil.
4. Place in thermocycler using the following routine:
   
   Thermocycler routine

   Step	Temperature (°C)	Time
   Initial Denaturation	98	30 sec
   35 cycles	98	5-10 sec
   66	10-30 sec
   72	20-30 sec/kb*
   Final extension	72	120 sec
   Hold	4-10

   *The recommended extension temperature is 72°C. Extension times are generally 20–30 seconds per kb for complex, genomic samples, but can be reduced to 10 seconds per kb for simple templates (plasmid, E. coli, etc.) or complex templates < 1 kb. Extension time can be increased to 40 seconds per kb for cDNA or long, complex templates, if necessary.
   **When amplifying products > 6 kb, it is often helpful to increase the extension time to 40–50 seconds/kb.
5. After thermocycling the product can be stored at -20°C or be used for gel electrophoresis.

By David Faurdal, adapted in part from NEB's one-taq protocol.
The purpose of this protocol is to confirm correct insertion of fragments after 3A assembly/GIBSON assembly into the pSB1C3 backbone. This is done by PCR amplifying the insert one is interested in and confirming that it's the correct size (by gel electrophoresis). As the backbone comes equipped with verification primer-binding sites, these primers, VF2/VR (sequences can be found here), can simply be used if one does insertion into it. The protocol can, however, be used with different primers for different backbones. As the fragments run on the gel won't be used for cloning purposes there is no reason to use high-fidelity polymerases on this, just use one-taq.

Materials

• Transformants from whatever assembly method you fashion
• Eppendorf tubes
• Sterile toothpicks/inoculation lops/pipette tips for transferring colonies
• Milli-Q water
• LB media with appropriate antibiotics

Procedure

Preparing the template DNA from the transformants

1. Pick a number of transformants, typically 3-10, from each plate of interest and mark them on the back of the plate.
2. Set up 2 Eppendorf tubes for each colony and mark them accordingly:
   1. Fill the first one (1) with 15 µl Milli-Q water.
   2. The other one (2) remains empty for now.
3. Transfer each colony to the Eppendorf containing 15 µl water using a sterile toothpick, inoculation loop or autoclaved pipette tip.
4. Transfer 5 µl of the water from (1) to the empty (2) tube. This tube (2) is now for safekeeping in case the colony PCR shows that the transformant in question contains the correct insertion.
5. Boil the (1) tubes for 10 minutes at 98 °C. Prepare the PCR mastermix, while the colonies are boiling.

Setting up the PCR itself

1. Set up a 25 µl reaction for each colony to be screened, as per NEB'S one-taq PCR protocol (see the protocol for troubleshooting).

   Component	25 µl reaction
   5x OneTaq Standard Reaction Buffer	5 µl
   10 mM dNTP	0,5 µl
   10 µM Forward Primer	0,5 µl
   10 µM Reverse Primer	0,5 µl
   OneTaq  DNA Polymerase	0.125 µl
   Template	1 µl from the (1) tube
   Nuclease-Free Water	up to 25 µl

2. Run the PCR in the thermocycler (use this website to calculate temperatures used based upon the primers and polymerase used).
3. Run the products on a gel to check for correct insertion.
4. Prepare an O/N culture from the (2) tubes that have the correct insertions by adding 1 ml LB media to the tube, mixing it and transfer a W-tube containing 4 ml LB media.

Adapted by Jacob Mejlsted

Materials

Consumables

• Ν 2 mL Fastprep tubes
• Ν 1.5 mL microcentrifuge/Eppendorf tube

Chemicals

• Lysis buffer or breaking buffer + LiAc
• Small glass beads
• 5 M NaCl
• Icecold 96% ethanol
• Milli-Q water

Instruments

• FastPrep machine
• Table centrifuge for 1.5 mL microcentrifuge/Eppendorf tubes.
• Heating block

Procedure

gDNA purification

1. Mix the following in a Fastprep tube: Scrape from plate colony and 500 µL lysis buffer or 500 µL breaking buffer + LiAc and 200 µL small glass beads.
2. Put in FastPrep machine at speed 4 for 40 seconds.
3. Spin down with a table centrifuge and transfer 150 µL of the supernantant to a new microcentrifuge tube.
4. Add 15 µL 5 M NaCl and 400 µL icecold 96% ethanol and mix
5. Spin 3 minutes at 10,000xg
6. Remove supernantant
7. Dry tubes on a heating block at 50 °C
8. Add 200 µL Milli-Q water and vortex
9. Optional: Spin down and transfer 150 µL to a new tube. (This can give a cleaner solution)