User:Jarle Pahr/Questions

Technical and scientific questions which can conceivable by answered by in silico or small-scale, low-cost wetlab experiments:

DNA purification/isolation:

How does additional washing steps affect the purity of miniprep DNA samples?

What is the difference and variation in yield for 6 mL cultures grown in 13 mL tubes and ErlenMeyer bottles, respectively?

How long must cultures be grown to achieve acceptable plasmid yields? Is 12 h enough?

Experiment: Inoculate several typical miniprep-scale cultures, incubate 12 h, perform miniprep, observe yields.

Ligation:

How does the probability of insert concatenation vary with insert concentration/molar amount (see Simulations)?

Transformation:

What is the smallest amount of plasmid DNA that reliably yield transformants?

What effect does snap-freezing have on the transformation efficiency of supercompetent cells? Experiment: Make one batch with half snap-freezed, the other half not snap-freezed, determine transformation efficienies for both conditions.

What variable (pre heatshock-incubation, post heat shock-incubation) has the largest effect on transformation efficiency (and how large is the effect)?

How does CFU vary with post heat shock incubation time?

Is pH adjustment (to 6.5) important for effectiveness of TSS?

SLIC:

Can small (~100 bp fragments) be succesfully cloned by SLIC? How? Suggestions: Use klenow polymerase, or very short incubation times. (add dCTP after 0 seconds, 10 s, 30 s, etc.). Klenow fragment has both 3'-5' exonuclease and 5'-'3 polymerase activity, so stopping the reaction with dCTP should work same as with T4 DNA polymerase.

PCR:

How does PCR yield vary with annealing temperature for a given reaction?

Experiment: Set up a gradient PCR and observe band intensity for the various positions. Or run several subsequent reactions with different annealing temperatures.

Misc:

How large must the production/concentration of GFP be for fluorescence to be visible to the naked eye upon illumination with UV light?

Non-standard growth media: Can E. coli grow on "dextro" energy tablets?

Can a defined medium for M. florum be developed?

Verification of commercial personal-genomics results: Can DNA extraction, PCR and sequencing confirm a SNP result reported by 23andme?

Commercial kit performance: What are the performance properties of various commercial molecular-biology kits?

Can horse meat be reliably detected by a cheap, at-home PCR test?

Do dNTPs show up during gel electrophoresis?

pllD: Can activity of the pllD promoter be shown by expression of GFP in a minimal medium?

What is the buffering capacity of LB?

Given a certain mutation rate, for how long can one expect maintain 100 % sequence preservation in a plasmid. When working with plasmids, a very large number of DNA molecules is handled simultaneously, but mutations occur in discrete molecules. What are the "population dynamics" of mutations?

What organisms live in a home environment? Can culturing, PCR and 16S sequencing be used to identify those organisms?

Does molten agarose contract or expand when solidifying?

LA medium darkens when stored in liquid form (warm) before pouring. What is the cause of this? Does it affect the nutritional properties of the medium?

Culturing and bacterial growth:

Does the initial cell density upon inoculation affect the final cell density?

How long does it take to reach maximal cell density for typical miniprep culturing ( ca. 3 mL in 13 mL tube, for example)

How long is a plasmid typically retained in the absence of selection pressure?

What is the maximal cell density (measured by OD600) for E. coli in LB medium (not pH adjusted) under typical miniprep culturing conditions?

What is the difference in growth rate and maximal cell density in LB using 5 g NaCl/L (Lennox LB) and 10 g NaCl/L, respectively?

Does cell density differ between cultures with same optical density but in different media (ex LB and M9)?

Can OD600 measured in a standard cuvette linearly related to OD600 measured in a microplate?

Storage:

How viable is storing bacteria (E. coli) at -20 C?

Antibiotics:

How stable is Kanamycin in solution at room temperature?

What is the stability of kanamycin with respect to repeated freeze-thaw cycles?

How stable is chloramphenicol in ethanol at room temperature?

Pipetting:

What is the effect of changing pipette tip versus re-using the same pipette tip when pipetting cell cultures of various densities?

What is the average volume of a drop from a drop counter (plastic pipette)?

Fluorescence:

How does sample volume (100 uL, 150 uL, 200 uL?) in microplate wells affect the results of microplate experiments? Which sample volume gives highest/lowest evaporation of sample volume as proportion of the original volume, when performing incubation experiments?

Bioinformatics/systems biology:

Can data from HTS be used to predict metaolic disorders?

• Call variants in the exome (known genes)
• For every variant, determine if it is a non-sense or mis-sense mutation.
• For non-sense/mis-sense mutations, consider if it is likely to affect protein function
• For putatively deleterious mutations, consider if loss of gene function is likely to affect the metabolic network in a medically significant way. (assess effect of gene loss on RECON model)

Test of concept:

• Find a well-described metabolic disorder in OMIM with known genetic cause
• Assess effect of loss of the causative gene in RECON

• Suggested test case: Phenylkeonuria (PKU). See Metabolic_disorders

Is there any added value from this approach?

• Known metabolic disorders with identified genetic cause can already be screened for without using metabolic modelling.
• For disorders which are unknown/previously unseen, no certain knowledge on the effect of mutations is available. (Not possible to reliably predict phenotype from genotype at this point).
• What is the probability that onset of a previously unknown metabolic disorder could be predicted from DNA sequence and metabolic modelling alone, before onset of symptoms? Seems unlikely/hard.

Added value would seem limited to an unlikely scenario where both:

• A: A metabolic disorder could be reliably predicted from DNA sequence and metabolic modelling alonge, before onset of symptoms.
• B: An effective treatment could be made based on knowledge of the disorder.

Conclusion: Metabolic modelling based on exome data seems unlikely to be an effective strategy at this point.

Big questions

Oligonucleotide synthesis: Will there be further radical improvement?

Is Cambrian Geonomics it?

Can enzymes be modified to emit a signal when a reaction is catalyzed (for studying metabolic fluxes, etc.)?

• See FRET nanosensors, discussed in http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3000467/
• See also "DNA-Based Sensor for Real-Time Measurement of the Enzymatic Activity of Human Topoisomerase ": http://www.mdpi.com/1424-8220/13/4/4017

Why does culturing fail for the vast majority of organisms? See http://schaechter.asmblog.org/schaechter/2010/07/the-uncultured-bacteria.html

Open questions in biology: http://www.biomedcentral.com/bmcbiol/series/openquestionsinbiology