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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

Introduction

Input amount of DNA to be purified should not exceed the binding capacity of the column (5 μg). A starting sample volume of 20–100 μl is recommended. For smaller samples, TE can be used to adjust the volume to the recommended volume range. Centrifugation should be carried out at 16,000 x g in a standard laboratory microcentrifuge at room temperature.

Materials

• Monarch® PCR & DNA Cleanup Kit
• DNA samples from PCR reaction

Procedure

Cleanup Procedure

1. Add ethanol to Monarch DNA Wash Buffer prior to use (4 volumes of ≥ 95% ethanol per volume of Monarch DNA Wash Buffer).

1. Dilute sample with DNA Cleanup Binding Buffer according to the table below. Mix well by pipetting up and down or flicking the tube. Do not vortex. A starting sample should include combined products of PCR amplification, i.e. 72uL for GFP15b/GFP27b/15b/27b