Team:CLSB-UK/Experiments

Experiments

Methodology

Protocols

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Amplifying gBlocks using PCR

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
Resuspension volume of TE buffer (μL)
gBlock gene fragment final concentration 15b/27b toehold switch (250ng) GFP switch (1000ng)
10 ngμL 25 n/a
20 ngμL n/a 50

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.
‘’’Component’’’ ‘’’Volume for the 25μL reaction (μL)’’’ ‘’’Volume for the 25μL reaction (μL)’’’
‘’’GFP’’’ ‘’’Basic blocks’’’
‘’’Q5 High-Fidelity 2X Master Mix’’’ 12.5 12.5
‘’’10uM forward primer’’’ 1.25 1.25
‘’’10uM reverse primer’’’ 1.25 1.25
‘’’Template DNA (1ng)’’’ 1 1
‘’’Nuclease-free water’’’ 9 9
  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).
‘’’Step’’’ ‘’’Temperature (℃)’’’ ‘’’Time (s)’’’
‘’’Initial denaturing’’’ 98 30
‘’’25 cycles’’’ 98 10
62 20
72 30
‘’’Final extension’’’ 72 300
‘’’Hold’’’ 4 Indefinite

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