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Introduction

gBlock Gene Fragments are normalised to 250 or 1000 ng, depending on length, which is a suitable quantity for many applications. However, we want to amplify gBlocks Gene Fragments to get additional starting material.

• GFP/ Luciferase 15b/27b - 1000ng
• 15b/27b - 250ng

Materials

gBlock Gene Fragments Amplification primers Phusion DNA Polymerase (www.NEB.com/ Phusion) PCR or gel purification kit (depending on application)

Procedure

Resuspening gBlocks

1. The dried down gBlocks Gene Fragment pellet can become displaced from the bottom of the tube during shipping. Centrifuge the tube for 3−5 sec at a minimum of 3000 x g to pellet the material to the bottom of the tube.
2. Add TE to the tube for your desired final concentration
3. Briefly vortex and centrifuge
4. Store at -20°C for up to 24 months

Resuspending the primers for PCR

1. Centrifuge the tube for 3−5 seconds at a minimum of 3000 x g to pellet the material to the bottom of the tube.

1. To make a 100 μM concentration: Take the number of nmoles of oligo in the tube and multiply that by 10. This number will be the volume (μL) of buffer to add to get a 100 μM solution.

1. Store resuspended oligonucleotides as several small aliquots at –20°C.

Amplifying gBlocks

1. A precipitate (most noticeable after the first 1–2 freeze/thaw cycles) is not uncommon. To ensure optimal performance, the master mix should be thawed and resuspended prior to use. Stability testing using up to 15 freeze/thaw cycles has shown no negative effect on master mix performance.

1. Dilute the gBlocks 15b/27b by using 1uL of the stock and 9uL of the TEB buffer. For other blocks use 1μL of the block and 19uL of the TEB buffer. This will give us both in concentrations of 1 ng/μL. Dilute the primers 1:10 to get 10μM concentration.

1. Assemble all reaction components on ice and quickly transfer the reactions to a thermocycler preheated to the denaturation temperature (98°C). All components should be mixed prior to use.

1. Gently mix the reaction. Collect all liquid to the bottom of the tube by a quick spin if necessary.
2. Transfer PCR tubes to a thermocycler and begin thermocycling (Programme 226).

The temperatures and times which the IDT document suggested for the thermocycling procedure were not successful at first. Our yield was lower than expected and this was due to “unclean” amplification where there was lots of non-specific primer binding. In an attempt to overcome this, we tried many different temperatures and timings. The table above illustrates the combination which produced the best results and gave us the highest yield.

The primers we used for our PCR reactions were designed using a primer tool on the Benchling website.

Source: IDT

Materials

• Gel Box
• Gel Tray
• Comb
• 1% Agarose in 1X TAE
• SYBR Safe
• 100ml beaker
• Pipettes and pipette tips

Procedure

Preparing the Gel

1. Prepare 300ml of the TAE buffer by dissolving 6ml of the 50X concentrated buffer in 294ml of distilled water.

1. Add 0.5g of agarose powder into 5ml of the diluted buffer and heat whilst stirring continually until clear.

1. Pour into the gel mould and add the comb with a required number of wells.

1. Pipette in 3uL of SYBR Safe dye and use the pipette tip to mix it around.

Preparing the material for loading

1. Mix 3uL of the DNA sample with 2uL of the loading dye.

Running the gel

1. Add enough 1X TAE to fill the reservoirs at both ends of the gel box and cover the surface of the gel - the gel should be immersed. You should use the remaining TAE buffer from 300ml.

1. Load 1μL prepared ladder (+dye +loading buffer). The concentration is 100μg/ml, i.e. there will be a total of 0.1ug = 100ng of ladder in the well. The mass of ladder is important to know if you need to quantify your bands by comparison with the ladder bands.

‘’Load ladder in left-most lane.’’

1. Use 2 μL loading dye per 3 μL of sample.

1. Load samples left to right.

‘’The capacity of the 8 well, 1.5mm wide well is approximately 45 μL. The capacity of the 15 well, 1.5mm wide well is approximately 15 μL.’’

1. Place cover on gel box such that your samples will run towards the positive, red electrode. Make sure that the cables from the cover are connected to the power supply correctly.

1. Turn on the power supply and run your gel at ~85 V for 1 hr 20 mins (voltage and time values can vary). Check regularly to see how far the dye has moved and stop the gel when the dye has moved approximately 80% of the distance.

1. Verify that bubbles are rising from the electrodes once you start your gel to ensure your gel is running properly.

SOURCE: http://www.openwetware.org/wiki/Agarose_gel_electrophoresis

Single-temperature Double digests for gBlocks

Introduction

This protocol was set up whilst waiting to see if we managed to successfully isolate the white colonies from the previous ligations. As PstI could have been the problem, we decided to try cloning the gBlocks into the plasmid using the EcoRI and SpeI instead and this combination of enzymes proved more successful that EcoRI and PstI.

This is the Double Digest Protocol with EcoRI-HF and SpeI, using a common reaction and same incubation temperature for both enzymes.

Double Digest Recommendations for EcoRI-HF® + SpeI: Digest in NEBuffer 2.1 at 37°C. ‘’At least one enzyme has < 100% activity in this buffer, so additional units of enzyme and/or longer incubation time may be necessary.’’

Materials

• Synthesised gBlocks
• NEBuffer
• NEB Restriction Enzymes
• Nuclease Free Water

Procedure

Single temperature double digest reaction

1. The dried down gBlocks Gene Fragment pellet can become displaced from the bottom of the tube during shipping. Centrifuge the tube for 3−5 seconds at a minimum of 3000 x g to pellet the material to the bottom of the tube.

1. Add 20µL TE buffer to the tube for your 50ng/µL final concentration (for the basic blocks dissolve in 12µL of TE buffer to get the same concentration)

Alternatively to the first 2 steps, use the gBlocks from the PCR amplification reaction.

1. Briefly vortex and centrifuge

1. Set up the following reaction (total reaction volume 50 µl). Restriction enzymes should be added last.

• Recommended maximum of 1 µg of substrate per 10 units of enzyme.

1. Mix components by pipetting the reaction mixture up and down, or by "flicking" the reaction tube.

1. Quick ("touch") spin-down in a microcentrifuge. Do not vortex the reaction.

1. Incubate for 1 hour at the enzyme-specific appropriate temperature. Then heat inactivate at 80°C.

1. PCR cleanup at the end to remove the short fragments.

Single-temperature double digest of BBa_J04450

Introduction

We previously tried using a linearised plasmid backbone but were not successful at identifying correctly ligated constructs. This prompted us to use BBa_J04450.

We need to digest enough of the BBa_J04450 plasmid for 4 reactions as we have 4 parts.

This is the Double Digest Protocol with EcoRI-HF and PstI, using a common reaction and same incubation temperature for both enzymes.

Double Digest Recommendations for EcoRI-HF® + PstI: Digest in NEBuffer 2.1 at 37°C. ‘’At least one enzyme has < 100% activity in this buffer, so additional units of enzyme and/or longer incubation time may be necessary.’’

Materials

• DNA
• 2.1 NEBuffer
• NEB Restriction Enzymes
• Deionised Water

Before the procedure aliquot all the enzymes and buffer first to prevent freeze-thaw in the future.

• Red - EcoRI-HF
• Yellow - PstI
• Green - DpnI
• Blue - 2.1 NEBuffer

Procedure

Single-temperature double digest reaction

1. Set up the following reaction (total reaction volume 30 µl). Restriction enzymes should be added last.

• Recommended maximum of 1 µg of substrate per 10 units of enzyme.

1. Mix components by pipetting the reaction mixture up and down, or by "flicking" the reaction tube.

1. Quick ("touch") spin-down in a microcentrifuge. Do not vortex the reaction.

1. Incubate for 1 hour at the enzyme-specific appropriate temperature. Then heat inactivate at 80°C.

1. PCR cleanup the product to remove the short fragments.

SOURCE: https://www.protocols.io/view/Single-temperature-Double-Digest-imsuj5

Introduction

This was the protocol we used for the first set of gBlocks with constitutive promoters.

Materials

• 10X T4 DNA Ligase Reaction Buffer
• T4 DNA Ligase
• Vector DNA - from linearised plasmid digest
• Insert DNA - from gBlock digests
• Nuclease-free water

Procedure

Set-up the T4 DNA Ligase Reaction

‘’Note:’’ T4 DNA Ligase should be added last. The table shows a ligation using a molar ratio of 1:3 vector to insert for the indicated DNA sizes.

1. Thaw the T4 DNA Ligase Buffer and resuspend at room temperature.

‘’Tip: Aliquot the 10x buffer less concentrated so when thawing, the DTT gets soluble more easily.’’

1. Set up the following reaction in a microcentrifuge tube on ice:

1. Gently mix the reaction by pipetting up and down and microfuge briefly.

1. For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes.

1. Heat inactivate at 65°C for 10 minutes.

1. Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells. Use 25 μL DH5α cells, and add 2 μL of reaction mixture.

Ligation with T4 DNA Ligase and ara-gBlocks

Introduction

This was the protocol we used for the new set of gBlocks which had arabinose promoters. We decided to discontinue the use of Luciferase gBlocks at this point in time.

Materials

• 10X T4 DNA Ligase Reaction Buffer
• T4 DNA Ligase
• Vector DNA - from linearised plasmid digest
• Insert DNA - from gBlock digests
• Nuclease-free water

Procedure

Set-up the T4 DNA Ligase Reaction

Vector concentration - 231ng from the digestion reaction which underwent PCR cleanup into 16uL of nuclease free water, making the concentration 14.4ng/uL. To get 30ng we need 2.1 uL of this stock.

15b/27b concentration - from the gel 15ng/ul. Use 1uL per reaction.

GFP15/GFP27 concentration - we digested 300ng of gBlocks and this underwent PCR cleanup into 16uL giving approximate concentration of 18ng/uL. To get 90 ng, we need 6uL of the block.

‘’Note:’’ T4 DNA Ligase should be added last. The table shows a ligation using a molar ratio of 1:3 vector to insert for the indicated DNA sizes.

1. Thaw the T4 DNA Ligase Buffer and resuspend at room temperature.

‘’Tip: Aliquot the 10x buffer less concentrated so when thawing, the DTT gets soluble more easily.’’

1. Set up the following reaction in a microcentrifuge tube on ice:

1. Gently mix the reaction by pipetting up and down and microfuge briefly.

1. For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes.

1. Heat inactivate at 65°C for 10 minutes.

1. Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells. Use 25 μL DH5α cells, and add 2 μL of reaction mixture.

SOURCE: https://www.neb.com/protocols/1/01/01/dna-ligation-with-t4-dna-ligase-m0202