Janet B. Matsen:Lab Tips & Tricks

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I came to the Lidstrom Lab with zero molecular biology or synthetic biology experience. I have great appreciation for all of the resources put online by the OpenWetWare community and other websites, so I am hopeful that I can share some of what I have learned. Please contact me with questions, comments, and corrections.

This collection is largely advice given to me by The Amazing Justin Siegel.

DNA Magic

• purifying DNA on a gel column has ~ 90% loss. purifying DNA on a column (non-gel) has ~ 90% recovery. avoid gel purification. (Justin)
• Freeze 'N Squeeze Gel extraction...?
  • I'd like to try. I even have enough of a kit to try some, but I never have samples I feel like taking the risk on! The only person I know who does it (also the person who gave me a piece of a kit) swears by it.
• Purify gels on gel-specific kits. Purify PCR products on PCR product specific kits. The former does a less effective job removing primer dimers. (Justin)
  • I don't actually do this because I like only having 1 kit. Keeps things simple.
• Try annealing @ 55oC in PCR before anything else.

Minipreps

• Grow in TB, not LB. This along gives me orders of magnitude higher miniprep yields.
• Grow overnight cultures in liquid:air ratios of at least 1:4; increasing the culture volume seems like a good idea at first but the lower oxygen concentrations are detrimental to plasmid yields.
• Specific suggestions for amounts of culture grow pSB plasmids overnight before miniprepping:
  • pSB1A3 (high copy number): 2mL of TB is sufficient for miniprepping. 5 mL in a 15 mL test tube is great.
  • pSB3K3 (medium copy number): 5+ mL in green-capped test tubes (they are 25+ mL)
  • pSB4C5: I have only done pSB4C5 (low copy number) once I prepped from 50 mL TB (overnight in a 250 mL flask) and got a very pleasing 200 ng/uL in 50 uL. The absorbance at 230nm looked higher than usual but the DNA peak was good. Now I usually do 5-10 mL in a green-capped tube, but have never gotten 200 ng/uL!

PCR

• Optimizing PCR rxns:
  • Try the PCR with 55oC for annealing and 0% DMSO first, almost always. If that doesn't work, you will see the manual gives you MANY options for things to change including template concentration, primer concentration, annealing temperature, Mg2+ concentrations, polymerase concentration, alternate buffers, extension time, and annealing temperature. There is no way you could test all the combinatorial possibilities here. I generally only change the annealing temperature and % DMSO if my template concentration is within the suggested range.
    • When Tm = 55oC failed, I used to do 12 rxns (20 uL each) with 4 temperatures (on a gradient PCR block), each at 4 temperatures ranging from 55oC - 70oC, and 0%, 5%, and 10% DMSO. If one of these conditions don't work, there is little hope that changing other things will be a game changer. Now that feels like too much work & I only do a few temps w/o DMSO.

Gels

• You can re-use tips when loading into wells if you rinse in the TB buffer in your block between every step. This is great because tips are ~$40/box. Don't do this for important samples (just in case.)
• If you are gel-purifing large volumes of PCR product, you can tape the lanes together. The greatest dangers are (a) the well ripping and allowing your sample to leak out and (b) having the buffer slosh you sample out of the well when you move or bump the box. Come talk to me about extremes in taping because I have made all the possible mistakes.
  • I don't actually do this any more (2012/07/02) because I am using Gibson cloning and don't need much DNA any more.
• Running large volumes on a gel ("overloading" the gel) causes the band size to run too fast. If you are running a large amount for purification, include a lane next to the ladder that is not overloaded so you can actually determine how big the bands are.

include small not-overloaded lanes when running purification gels

Equipment

• Check out my rebound board for discarding used tips! I won't be shooting tips onto Amanda's bench any more.

tip back board

Media

• TB (terrific broth) gives you healthier cells and more plasmids than LB. I use LB for freezer stocks because or media prep person makes it in abundance but TB for minipreps to get higher plasmid yield.

Transformations

• Plate transformations by spreading with a bent 20 uL pipette tip, not an ethanol/flame sterilized stick, glass beads, etc. You have to use the pipette tip anyway to slurp up some of the cells, so just bend it using the inside of the eppendorf tube and use it to spread the cells.
  • I have light a small fire using a flame-sterilized stick, and roller beads are a hassle and hard to get non-uniform spreading of cells with. I like nonuniform spreading because it will allow you to grab single colonies even if your transformation efficiency is really high or really low.

nonuniform plating of transformed cells is good

• Don't transform plates at room temp over the weekend, especially if you rely on Amp resistance. Often you see a lot of contamination! (Janet)

Keeping primers, plasmids, strain stocks, & sequencing organized

• Keep a master google spreadsheet with a page each for primers, plasmids, strain stocks, and sequencing. (example) This will keep you very organized, and allow you to collaborate more easily.
  • On the primers page, each primer should have an individual number, a name without spaces, the sequence, comments for its intended use, and the date you ordered it. When ordering primers from IDT, you can paste the three consecutive columns with primer number, name, & sequence into the multiple primer entry window. Then delete the space between the primer number & name, and add a hyphen. You can grey out primers you decide are no longer useful (example: you find out it is wrong or doesn't work).
  • On the plasmids page...
  • On the strain stocks page...
  • Keep the primers, plasmid stocks, cell stocks, and strain stocks in boxes with their unique identification number on top. Order them by number. This allows for ease of sharing resources & information. Note that the plasmids can be stored as minipreps and in the cloning strains, so you will have two boxes that share the same numbers.
• Keep a dropbox folder with the final versions of all of the plasmids you trust & keep.
  • I name them by their plasmid number & short description.
  • In this, I keep an extra copy of the sequence file that is annotate with all of the confirming sequencing rxns I did. For each sequence that comes back, I add a feature that is named with the order number of the sequencing reaction & the primer that was used to obtain the sequence. The feature should cover only the part of the sequence for which the sequencing reaction was solid. This means visually trimming off areas with N's.

Misc

• Don't be surprised if you get contamination on plates with multiple antibiotic resistances. There are plenty of plasmids floating around natural systems with multiple on them already. Bacteria make antibiotics in natural systems to add competition to an an environment. (Mary Lidstrom, 2012)