Day 7 Morning Lecture Notes

Steve Williams, Smith College

June 12, 2004

RNA and DNA form stronger bonds to each other than DNA-DNA bonding. Primers used in eukaryotic DNA replication are also RNAs. Two strands of RNA can form a double helix. Some viruses have double-stranded RNA genomes.

How much insert and vector do we want to use in a ligation reaction? Ideally (ng insert/MW insert)/(ng vector/MW vector) = 7.5. We used 25 ng of a 3850 bp vector, so for a 1600 bp RevT insert, we need about 80 ng of insert to get the optimal ratio. We are able to use a high insert/vector ratio since the inserts cannot bind to each other due to their having an EcoR1 overhang on one end and an Xho overhang on the other.

Sources of PCR Contamination

A genomics lab that works with mouse is liable to be contaminated with mouse DNA, RNA, cDNA, genes cloned in lambda, genes cloned in plasmids, and previous PCR products. The latter are the biggest problem. Because DNA is so stable, it's on all lab surfaces at all times. A central facility lab that works with many species has fewer concerns about contamination.

Every time a microfuge tube is opened, a contaminating aerosol is generated. Puling aerosols into unfiltered tips is also a problem.

Viruses with lipid polysaccharide coats on their surface are destroyed by air. Those with protein coats like lambda are air-stable.

Common steps to help reduce contamination in PCR reactions
(in order of increasing difficulty and expense; see also p. 123-125 of handouts)

1. Always wear gloves and change them frequently.
2. Use filter-barrier pipet tips.
3. Use a dedicated set of pipets for the PCR setup. Never use pipets that were used with PCR products to set up a new PCR reaction.
4. Have a dedicated room or bench for PCR prep and handle PCR products elsewhere.
5. Clean lab benches, equipment and pipets frequently with a 5% bleach solution from a spray bottle. Bleach causes DNA to cross-link.
6. Aliquot PCR reagents to each individual investigator. That way contamination sources can be identified and contained.
7. Add template to PCR reactions last. Mix all other reagents first and put the containers away. Then bring out the template and add it to the mix. Or at least store the template at the other end of the bench from the reagents.
8. Use uracil n-glycosylase (UNG). How does this work?

UNG in cells removes U's from DNA and (in the presence of dTTP) replaces them with T's. A U-containing gene cloned into a vector will have them replaced by T's. In vitro UNG only removes U's, fragmenting the DNA.

Suppose we run the initial reverse transcription step of RT-PCR with dTTP but use dUTP for all subsequent PCR amplifications. Any contaminating product from a previous PCR reaction is therefore damaged by UNG. The real cDNA template from the reverse transcription reaction will not be affected by the UNG. This UNG trick was developed by Applied Biosystems. However any contamination with genomic DNA will still contain T's and can still be amplified.

9. Use a tissue-culture hood to mix reagents. These hoods blow sterile air down out of the hood. They also have UV lights for cross-linking that must be turned off when template is handled (!). Similar bench-top PCR hoods are available. Usually these hoods are overkill.
10. Special RNAse sprays and wipes are available, but once again these are usually overkill.