Janet B. Matsen:Lab Tips & Tricks

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*Optimizing PCR rxns:
*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.  I generally 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.   
**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.  I generally 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.   
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===Gels===
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*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.
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*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.
==Media==
==Media==

Revision as of 17:22, 2 March 2012

Back to Janet

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.

Contents

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...?
  • 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)
  • 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 .
    • pSB3K3 (medium copy number): 2*2mL (parallel culture tubes) of pSB3K3 is sufficient but 3*2 mL is better.
    • pSB4C5: I have only done pSB4C5 (low copy number) once but I prepped 50 mL from an overnight culture in a 250 mL flask and got 50 uL with 200 ng/uL and was pleased with it. The absorbance at 230nm looked higher than usual but the DNA peak was good.

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. I generally 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.

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.

Media

  • LB gives you more cells than TB/
  • Use TB (1-2 mL) overnight for plasmid preps (for pSB1A3, pSB3K3)

Transformations

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)
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