User:Jarle Pahr/lablessons

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13.02.13: Phusion PCR raw product is unsuitable for nanodropping (viscosity issues). True in general for raw PCR products?
13.02.13: Phusion PCR raw product is unsuitable for nanodropping (viscosity issues). True in general for raw PCR products?
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13.02.13: Phusion PCR raw product can be loaded directly on gel without loading dye.
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14.02.13: Don't work late. Better to go home and continue early next morning.

Revision as of 06:31, 14 February 2013

01.02.13: LA medium does not solidify before it is heated once.

01.02.13: If cap is screwed tightly on, liquid in container may "boil over" when container is opened after autoclaving.

01.02.13: Time for 500 mL LA medium in 1000 mL Schott bottle to cool from ~55 C to a temperature that is not painfull, when the wrist is touched to the bottle (temp when it's OK to add antibiotic): ~40 min

01.02.13: Time for LA plates to solidify: < 20 min

01.02.13: Time for agarose gel to solidify: 30-40 min (?).

01.02.13: Commonly used Kan concentration on LA plates: 25-50 ug/mL

02.02:13: If toothpicks are used for inoculation instead of an inoculating needle, there's no danger of inoculating the same sample twice by mistake.

03.02.13: Don't tighten the centrifuge rotor cover too tightly. If it's hard to hopen, a forceps can be used to gain leverage.

03.02.13: BSA does not affect the activity of enzymes which don't need it for stabilization (http://en.wikipedia.org/wiki/Bovine_serum_albumin)

03.02.13: PciI and AgeI are not Dam sensitive.

03.02.13: Recommended restriction digest incubation time may vary from 1 (diagnostic digest) to 4+ (cloning, >1 ug DNA) h. (http://www.addgene.org/plasmid_protocols/restriction_digest/)

05.02.13: Effects from loading dye may cause mismatch between sample DNA bands and DNA ladder bands.

05.02.13: When incubating large cultures, fill up maximally 20 % of the vessel volume. Ex, for a 1000 mL ErlenMeyer bottle, use maximally 200 mL medium.


http://bitesizebio.com/articles/a-beginners-guide-to-storing-biological-materials/


12.02.13: Do everything twice.

12.02.13: 6 ng/uL is a *good* yield for agarose gel extraction, according to Rahmi. 100 ng should be plenty for SLIC.

12.02.13: For Phusion polymerase, annealing temp should be set to 3 C above lowest-melting primer IF the primers are longer than 20 nt. If the melting temperature is particularly high (ex, 65), then it's OK to use the melt temperature directly.

13.02.13: Keep track of samples! When receiving samples, make a note and confirm the correct number of samples, then immediately place the materials in their long-term storage location.

13.02.13: Keep PCR tubes in Eppendorf tubes when not in the PCR machine.

13.02.13: Too high primer concentration can inhibit DNA polymerase, according to Rahmi.

13.02.13: PCR tubes can be centrifuged using a holder/adapter.

13.02.13: Time for full enzyme/buffer tube to thaw from -20 C:

13.02.13: When running many PCR samples at the same time, it is convenient to make a master mix.

13.02.13: PCR tubes may deform if left at 95 for a prolonged time.

13.02.13: Phusion PCR raw product is unsuitable for nanodropping (viscosity issues). True in general for raw PCR products?

13.02.13: Phusion PCR raw product can be loaded directly on gel without loading dye.

14.02.13: Don't work late. Better to go home and continue early next morning.

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