DNA synthesis and primers

DNA synthesis

We always get primers from IDT and genes/longer DNA fragments from Twist. If it's less expensive or there's another good reason, sometimes we buy genes at IDT too.

Ask someone in the lab for the login and password information for our accounts.

• Gene fragments at Twist
  • Gene Fragment with Adapters (300bp-500bp) - $35.00 flat fee
  • Genes with or without Adapters (501bp-1.8kb) - $0.06/bp
  • Genes with or without Adapters (1.8kb-3.2kb) - $0.06/bp
  • Genes with or without Adapters (3.2kb-5.0kb) - $0.08/bp
  • Gene without Adapters (3.2bp-5.0kb) - $0.09/bp

At Twist, we also have discounts on clonal genes (genes cloned into one of their, or our, vectors); custom vector onboarding; and oligo pools. And we don't pay shipping.

• eBlocks (IDT)
  • 300 to 900 bp
  • need to order at least 24 sequences in a plate
  • OK quality
  • 1-3 business days
• gBlocks (IDT)
  • Can order longer sequences in tubes or plates
  • These take longer to be delivered and are also higher quality
  • ~2 weeks for a 1 kb gene

Primers

• 60 bp max for 25 nM concentration - these take 1-2 days
• General guidelines for primer design
  • for standard PCRs, aim for a Tm of 50-60 °C and not higher than 71 °C
  • make the Tm match for primers that are to be used together
  • if the primer Tms are quite different, try a touchdown protocol
  • check to see what the effective Tm is for your polymerase
  • try to end the primer on a G or C
  • for primers and/or genes that have high GC content, try adding 1% DMSO to the reaction, or using Phusion polymerase with the high-GC buffer
  • anticipate mispriming, and extend the primer if it might be a problem