Cloning Checklist

E. Coli Cloning Checklist

This is a rough guide to the DOs and DO NOTs of cloning in E. Coli. It was assembled based on problems I've encountered while attempting restriction digests, ligations, and PCR.

Overall Plan

• DO create/destroy restriction sites.
  • This will allow for better verification of what you've done. It's just easier to tell the difference between the number of bands than the size of bands.
• DO plan validation digests from the beginning.
  • If you plan these while you're choosing enzymes, you can avoid having to try to tell the difference between 1300bp and 1350bp. You can have less difference between low molecular weight fragments (~100 bp if fragments are < 1kb). However, high molecular weight fragments require large differences (sometimes more than 2 or 3 kb if <3 kb). If you can clearly tell the difference between two lenghts on your ladder, it will probably work for a digest, too.
• DO take advantage of mutagenesis.
  • Mutagenesis is simple and reliable and can save LOTS of time. Use it to switch up restriction sites throughout the vector. But don't forget, silent mutations aren't always silent, they effect translation and folding kinetics, so try to make silent mutations that have similar codon bias. OpenWetWare has a good codon bias table.
• DO NOT interfere with upstream regulatory regions.
  • Unless you mean to change them, too. Promoters have important regions about 35bp and 10bp before the start codon. And don't forget about the Shine-Dalgarno sequence (consensus in E. Coli: AGGAGGA) which is needed for mRNA to find the ribosome.
• DO gel purify after restriction reactions.
  • You'll lose more DNA than with a PCR cleanup kit, but you're more likely to get what you want if you get rid of the DNA pieces you don't want.
• DO NOT change the reading frame of the protein.
  • Make sure to translate your final product before ordering any primers. This will make sure you kept the right reading frame throughout your construct.

Choosing Restriction Enzymes

• DO check for enzyme uniqueness in both the backbone and insert DNA.
  • It's simple, but a terrible thing if you forget to... Restriction sites can be created/destroyed before cloning by mutagenesis.
• DO NOT create blunt ends.
  • Technically they work. Practically, not so well... Cloning will go much smoother with sticky ends, so avoid blunt ends if possible. Even doing a mutation to add a sticky restriction site will probably save you time.
• DO use enzymes with different sticky ends.
  • This will allow you to make sure your DNA ends up in the vector in the right direction. It will also not allow the backbone to close on itself (can be fixed by dephosphorlyating the 5' end of the backbone).
• DO check for methylation sites.
  • dam methyltransferase: GA*TC
  • dcm methyltransferase: CC*AGG, CC*TGG
  • Methylation inhibits restriction enzymes. NEB lists dam or dcm sensitivity for each of their enzymes. If your enzyme is on this list, make sure there is no overlapping methylation site. If there is, fix it by mutagenesis or do your cloning in dam-/dcm- E. Coli strains (sold by NEB).

PCR

• DO include 8-10 random nucleotides between the end of the PCR fragment and a restriction site.
  • Restriction enzymes need DNA to 'sit' on so that they can cut. If the restriction site is too close to the end, the enzyme will fall off the DNA before it's enzymatic activity can occur. My favorite filler is CATGTAGC.
• DO make sure your primer's 3' nucleotide is G or C.
  • G and C bind tighter than A and T. Ending with G/C gives the polymerase a good base to start polymerization.
• DO NOT make the primer too G/C rich.
  • This will make the primer 'sticky' and lots of nonspecific binding/polymerization will occur. 50% is a good G/C target. This is especially important at the 3' end of the molecule.
• DO make sure your primers have similar [math]\displaystyle{ T_m }[/math]s.
  • They should be within 5°C of each other. Set your PCR annealing temperature to 5°C less than your lowest [math]\displaystyle{ T_m }[/math]. Only calculate the [math]\displaystyle{ T_m }[/math] for the sections which anneal.