User:Jarle Pahr/lablessons

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. Or use centrifuge PCR tube holders.

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.

15.02.13: SLIC and Gibson assembly are related methods. The starting materials and final products are the same (the primers are compatible). SLIC primers might also be used for RF or CPEC cloning..

15.02.13: Fragments for use in SLIC should be at least 250 nt long to avoid complete degradation by exonuclease activity of T4 DNA polymerase.

17.02.13: pH meters will not give correct readings in distilled/deionized water. (http://www.vernier.com/til/1286/)

17.02.13: DNA is not stable in the *long term* at >2 C in H20. http://nucleicacids.bitesizebio.com/articles/what-actually-happens-if-you-store-dna-in-water/ TE buffer stabilizes DNA by preventing hydrolization.

18.02.13: Plan/prepare experiments before going to the lab.

18.02.13: Loading dye and DNA samples may be mixed in a droplet on parafilm.

18.02.13: Restriction digests can be loaded (at least in small amounts, 2 uL) directly on gel without loading dye and give a useful image.

18.02.13: dNTPs may degrade after several freeze-thaw cycles. dNTPs should be stored at at least 10 mM concentration to decrease degradation by hydrolysis. dNTPs stocks from different manufacturers may have different stabilities. (Some sell "room temperature stable dNTPs". http://www.webscientific.co.uk/acatalog/Room_Temperature_Stable_dNTPs.html, http://www.thermoscientificbio.com/general-reagents-and-accessories/dntp-set/) Fore more info on dNTPs, see http://www.amplifa.com/en/products/dna-technique/faq-dntps-pcr/

18.02.13: EDTA is an PCR inhibitor.

18.02.13: When pipetting, keep a close eye on the pipette tip to make sure the desired amount is actually transferred.

18.02.13: When pipetting phusion enzyme solution (glycerol based? May apply to glycerol solutions in general), solution may enter the pipette tip even if the plunger is not released. -> May give larger volume than desired.

18.02.13: Phusion polymerase does not have strand displacement activity, and can be used for RF cloning.

18.02.13: From Bryksin and Matsamura 2010:

"In general, PCR yields are poor when the reaction conditions are too stringent (primers fail to anneal) or too relaxed (nonspecific priming). Both are manifested by empty lanes in agarose gels, although the latter can also result in smears or undesired bands"

18.02.13: To allow inspection early next morning (16 h incubation time), transformations should be completed no later than 16 PM.

18.02.13: Time for 0.1 mL supercompetent cells to thaw on ice:

19.02.13: dpnI is a restriction enzyme which cleaves A-methylated GATC sites. pSB-M1g has 34 dpnI sites.

19.02.13: Plan construct verification and test cutting in a systematic way. Aim for a solution where all constructs can be verified in a standard way (using the same restriction enzyme(s), etc.).

19.02.13: When cutting a sequence with a restriction enzyme, the number of fragments obtained will be different depending on if the sequence is circular or linear.

19.02.13: Cutting a circular plasmid at N sites will result in N fragments. Cutting a linear DNA molecule at N sites will result in N + 1 fragments.