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Revision as of 06:54, 25 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.
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.
19.02.13: The restriction sites most likely to be present in an insert are also most likely to be present in the vector.
19.02.13: Centrifuge newly arrived primer tubes before opening, to avoid loss of DNA.
19.02.13: After dissolving primers, verify/control DNA concentration with NanoDrop.
19.02.13: On NanoDrop, use ssDNA-33 option to measure single-stranded DNA such as primers.
19.02.13: When using NanoDrop, don't just look at the numbers. Also look at the graph! In particular, where is the absorbtion peak? Save results for all important samples, including the graph!
19.02.13: When measuring high DNA concentrations on NanoDrop, significant amounts of DNA may carry over to the next sample if the pedestal is not thoroughly cleaned. Example: Primer DNA measured to -> 1554 ng/uL (ssDNA), next sample MQ water measured to 128. ng/uL. -> Next sample: 48 ng/uL. Need to be find a reliable protocol for cleaning the pedestal.
20.02.13: The only difference between CPEC and RF cloning is that in CPEC, a linearized (cut) plasmid is used, while in RF, an intact plasmid is used, followed by digestion of the original plasmid with DpnI.
20.02.13: The RF cloning procedure can be used for either REPLACING a sequence in the original plasmid, or for INSERTING an additional sequence into the original plasmid WITHOUT removing any of the original plasmid sequence. If an RF megaprimer is to be used to REPLACE a sequence, the insert should be about the same size as the sequence to be replaced. Source: ?
21.02.13: When making SLIC primers, make sure to check for stable secondary structures.
21.02.13: Read protocols carefully. Otherwise, you might forget the isopropanol...Update: When using kits, ALWAYS review the protocol carefully, so you don't forget any steps.
21.02.13: T4 DNA polymerase has 200x 3-5 exonuclease activity compared to Klenow fragment. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC332407/pdf/nar00204-0127.pdf
How about using klenow fragment instead of T4 DNA polymerase for SLIC?
21.02.13: When performing gel extraction with QiaGen QIAquick kit, a peak may present at 230 nm, caused by Guanidium salts in QG buffer (http://seqanswers.com/forums/showthread.php?t=15165, http://www.protocol-online.org/biology-forums-2/posts/6994.html, http://www.protocol-online.org/biology-forums/posts/42395.html). Guanidium salt may inhibit enzyme activity! Should attempt to avoid this contamination. Suggestions: Wash several times with PE; incubate longer with wash buffer; heat wash buffer to 37 C.
From www.biochrom.co.uk/download/70/ :
"An elevated absorbance at 230 nm can indicate the presence of impurities as well; 230 nm is near the absorbance maximum of peptide bonds and also indicates buffer contamination since This, EDTA and other buffer salts absorb at this wavelength. When measuring RNA samples, the 260/230 ratio should be > 2.0; a ratio lower than this is generally indicative of contamination with guanidinium thiocyanate, a reagent commonly used in RNA purification and which absorbs over the 230 - 260 nm range. A wavelength scan of the nucleic acid is particularly useful for RNA samples."
At "http://www.protocol-online.org/biology-forums/posts/8553.html", "Bob" writes:
"Typically there isn't a peak at 260 for DNA, there is a rounded peak lower in the spectrum, at around 230. The DNA is measured at 260 as the 230 peak is not specific for DNA, however as this measurement is on the side of the 230 peak it can be quite variable."
Even without a peak at 260 nm, the DNA might still be useful.
22.02.13: When measuring DNA concentrations in a DNA sample, and blanking against the buffer, use buffer from the same bottle for eluting and blanking.
22.02.13: When excising gel fragments, bring a palm scale to weigh the gel pieces.