Project management and execution
A failure is not a failure.
If it's not recorded, it didn't happen.
The golden rule: Sequence, sequence, sequence! Don't trust what's been handed down to you, don't trust what you have made yourself too much either. Mutation happens. 'Always sequence at least two clones.
When planning a cloning project in a new vector, plan for and try several cloning strategies to see what works before committing to one method.
Perform all cloning reactions in silico first to avoid embarassing mistakes (like using a restriction enzyme having several cutting sites when you think there's only one). After receiving sequencing results, align the determined sequence against the theoretical sequence. Save expected plasmid sequences to align sequencing results against later. After saving a construct sequence, Check that all critical features (neccessary restriction sites, start and stop codons, etc.) possibly affected by the cloning are correct.
- First, BLAST (or otherwise align) each sample result to the expected theoretical sequence.
- If several good samples look good, perform multiple alignment.
- If "no significant similarity found", check that the most permissive algorithm (blastn or similar) is used. If still no similarity found, inspect the chromatogram.
Hurrying costs time! A mistake from a few seconds inattention can take hours to repair. Ex: Forgetting to add dNTPs to the PCR.
Consider employing a Checklist for routine operations to make sure nothing is forgotten.
Keep track of cloning progress (confirmed sequences, desired and completed constructs) in a spreadsheet or other document. Keep pick-up dates for sample submissions in mind when planning work.
Create files and folders containing to keep track of the following sequence categories:
- Plasmids (determined sequences/archived plasmid maps)
- Constructs (theoretical, desired sequences for new constructs)
- Functional sequences (Promoters, degradation tags, etc. Make one folder per category)
- PCR products (Expected sequences of PCR products)
- Restriction site sequences (with padding nucletoides. For appending to primers.)
Suggested template for folder and file structure:
- Cloning.doc (Planning of cloning)
- Constructs.doc (Tracking of planned and completed constructs)
- Oligomers.doc (Sequences of DNA oligomers to order)
- Samples.doc (Information on samples of all types, including PCR products, restriction digests, ligation mixtures, glycerol stocks and agar plates, : name, status, references, etc.)
- Bibliography.doc (Notes on relevant publications)
After obtaining cultures of candidate clones, consider - based on the number of cultures, number of colonies on the plates, etc. - if culture should be saved as glycerol stock. This guards against loss of the clone in case plasmid preparation fails or the plasmid DNA is later lost, and removes the need for re-transformating the plasmid DNA if the desired sequence is confirmed.
For verified samples, two separate glycerol stocks to be kept to guard against sample loss in case of contamination, loss of viability or other accident.
After receiving sequencing results, as soon as possible discard any clearly bad samples (re-ligations, etc.) to avoid cluttering of storage boxes. If several apparently identical (good) clones are obtained, consider how many should be retained.
If nucleotides are missing in sequencing result: Check primers.
Work and planning documents:
It may be useful to documents for the following, placed in their appropriate locations:
- Sequences of interest. To contain sequences of interest and notes on them.
- Oligomers. To contain proposed oligomer sequences.
- Orders. To contain the sequences of oligomers and sequences that are actually ordered.
- Constructs/progress sheet. To contain information on constructs and plasmids under construction.
- Samples: To contain information on intermediate and final products and samples (PCR products, Digests, ligations,, supercompetent cells, glycerol stocks etc.) with appropriate data (concentrations, transformation efficiences, etc.)
- Sequencing results. To contain notes on sequencing results.
- Gel results. To contain gel images and notes on them.
- Project plan: To describe the long-term, high level plan for the project at an overall level.
- Cloning: To describe cloning strategies. Include all relevant information.
- Working plan: To describe the near-term plan and/or longer-term plan at the specific level. Progress may be checked off against the goals in this plan.
- Daily notes: To contain miscellaneous daily notes.
Tip: Add information on samples to their respective documents as soon as possible after mixing the sample (while incubating the sample, for example).
Lab planning/organization platforms such as LabGuru may also be helpful.
In addition to electronic records, keep a ring binder with paper documents for the following in the lab:
- DNA concentration measurements
- Oligomer quality control sheets
- Material safety sheets
- Important articles
All files and documents which will be edited after creation, or are results, should preferably be contained in one top-level folder subject to version control. For example, store all such files in a folder named "dev".
Start every day by inspecting any experiments/cultures left overnight. Then review and if necessary revise the near-term work plan.
Logging of work: Based on complexity and reliability of procedures, consider which tasks which should always be logged in detail (writing down volumes added, etc.) and which can be considered to be "standard operating procedures".
WRITE THE DATE on top of all pages in the lab journal.
Suggested tasks that may be considered "SOP"
- Preparation of complex media (LB)
- Purification/clean-up of PCR products/digests
- Miniprep using kit
- Inoculation of liquid cultures of in standard media
For SOP's, it is enough to note in the log that the task was performed.
Tasks which should always be logged in detail:
- Setup of restriction digests, ligations and PCR reactions.
Measurements: When to measure, and what values are expected for measurements in various standard operations?
Some quality control can be sacrificed for time, by making the following assumptions with regards to typical measurment values:
- DNA concentration in purified restriction digest (~1 ug DNA in 20 uL, purified with Qiagen PCR purification kit and eluted in 50 uL EB):
Start of work week: Prepare consumables for the week (medium, pipette tips, etc.)
End of work week: Review analysis results produced/received during the week. Discard redundant/un-needed samples, and arrange/plan for duplication/storage of valuable samples.
Beginning of work day: Review what should be done today.
End of workday: Review what should be done the next day.
"Blind" cloning: If time is limited, or it is otherwise not possible to make concentration measurments, the following volumes can be used as a suggestion:
- Backbone: 10 uL (assuming a DNA concentration ~ 10-20 ng/uL
- Insert: 1 uL
You don't write the notebook for today, you write it for tomorrow!
28.02.13: Regularly clean the lab bench, to decrease the chance of contamination. Worst case, if the bench is contaminated with antibiotics, recombinant organisms could be selected for, increasing the chance of contamination even of selective media.
25.02.13: Being left-handed, it is best to place the pipette tip box on the right side of the bunsen burner when using sterile technique.
11.03.13: Keep a paper inventory of samples for quick reference.
Replacement of stock solutions:
To guard against contamination, consider if the following should be replaced weekly:
- LB / rich growth media
- sterile glycerol
Time estimates for various operations:
13.02.13: Time for full enzyme/buffer tube to thaw from -20 C:
18.02.13: Time for 0.1 mL supercompetent cells to thaw on ice: ~10 min
Time needed to pack 96-tip pipette tip box: ?
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: ~ 20 min (?).
Time for LA plate in plastic petri dish to warm from 4 C to 37 C when placed in 37 C incubator:
18.02.13: To allow inspection early next morning (16 h incubation time), transformations should be completed no later than 16 PM.
Time needed to purify restriction digests (~4), determine concentrations, and set up ligation reactions: 1 h
25.02.13: After pouring a gel, check that that the gel is level, by looking at the gel horizontally at eye-level (crouch down).
28.02.13: Use the time while the gel solidifies to mix samples and loading dye.
05.02.13: Effects from loading dye may cause mismatch between sample DNA bands and DNA ladder bands.
LA medium in a bottle may solidify at the top even though a magnetic stirrer at the bottom is spinning.
After preparing antibiotic-containing agar plates, inoculate one plate with a non-resistant strain and incubate overnight to test the antibiotic efffect.
To decrease chance and impact of contaminations, preferably prepare several smaller batches larger than one small. For example, 250 mL LB in 500 mL bottles is a suitable size.
Material/sample handling and storage
To decrease the chance of perpetuating contaminations, prefer inoculationg a new culture (from agar plate or glycerol stock) over passaging cultures.
When retrieving cells from glycerol stock, use a cooling block (enzyme cooler) to keep the cryotube cold and in a stable position.
In a freezer, cold air sinks to the bottom. If the freezer is empty, there is more air in the freezer, and the temperature difference between the top and bottom will be larger. Especially temperature sensitive samples such as supercompetent cells should be stored as low as possible in the freezer.
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.
07.03.13: MARK ALL TUBES WITH DATE.
When finished using a sample tube, make sure that the cap is completely closed. Otherwise, the sample may evaporate if left on the bench. If the cap is broken during centrifugation, transfer the sample to an intact tube.
To make keeping track of samples easier, the following method can be used: Favorite samples can be marked with a green marker dot on top of the tube. Samples to be discarded/samples with no useful content can be marked with a red dot.
Avoid potentially confusing names. For example, do not include "LIG" in plasmid sample names (as it may seem to imply a ligation mix), even if the plasmid was derived from a sample which was named as such.
As a general rule, glycerol stock should always be made when harvesting culture for DNA isolation. 2 minutes per sample can save a day(!) later (liquid culture can be inoculated directly from glycerol stock. If only plasmid DNA is available, cells must be transformed, plated and incubated overnight before transferring liquid culture).
If the culture volume available for harvesting is small, a portion of the miniprep culture can be used to inoculate another culture to use for preparing glycerol stock. Alternatively, a drop of each culture can be placed in separate, marked spots on an agar plate, and the plate incubated overnight. If restriction analysis / sequence analysis indicates a correct clone, a culture can then be inoculated from the corresponding spot on the plate.
To avoid loss of construct due to contamination or loss of viability due to use during a project, observe the following rule: When working with a given construct, in addition to glycerol stock, also maintain at least samples of either plasmid DNA or colonies on agar plate. Don't keep all your eggs in one basket!
Before long term storage, inoculate a culture from glycerol stock, isolate plasmid DNA and confirm that the correct construct is re-obtained, before considering the glycerol stock verified OK for long-term storage.
If possible, keep use two boxes (or separate parts of one box) for storing glycerol stock samples: One for temporary storage (un-verified samples), and one for verified samples.
The following nomenclature syntax can be used to systematically name samples:
- PROJECT/#parentsample[prefix1]#subsample[prefix2]#subsample2 etc...
Goal: The name of a sample should make it possible to trace the origin and history of the sample. All symbols should be easy to write and discern on tubes, etc. As much as possible avoid potential confusion with symbols which may appear in sample names. Ex: a plasmid named "pSB-M1g" makes using the "-" as a sample type prefix unadvisable.
Advantages: The history/origin of the sample is recorded in the name.
Disadvantages: Sample names may become long and unwieldy. However, names can be shortened by defining a new (that is, older) sample as the parent sample, only recording the sample history from that point onwards in the name. To preserve the entire sample history, it is then only necessary to preserve the full name for the parent sample. Decisions for which names to shorten could be based on sample hierarchy structure so that frequently used samples (or samples stemming from a frequently used sample) are given shorter names.
Sample type prefixes:
. : Plate
\ : Liquid culture
": " glycerol stock
l : ligation
> : Isolated DNA
^ : Other sample
Agar plate of pSB-M1g from glycerol stock, "Master" project.
Same as above, but without specifying origin of sample (as glycerol stock).
.pSB-M1g\A>1> Isolated plasmid DNA sample 1 from liquid culture A from pSB-M1g agar plate.
Simplest nomenclature: Sequential numbering. "p(Initials)-(number)" Advantages: Simple, unambigious. Easy to write on physical samples. Disadvantages: Non-descriptive. Numbers may run high.
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: 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. An empty pipette tip rack can also be used.
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: 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"
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.
22.02.13: A 10 uL pipette tip rack can be used to hold PCR tubes.
22.02.13: When doing standardized PCR protocols (same PCR program for all samples), if time allows also do a control reaction known to work, to check that enzyme and dNTP (and template) are OK.
25.02.13: T4 DNA polymerase has 60 % activity in NEBuffer 1, 100 % activity in NEBuffers 2-4. (https://www.neb.com/products/M0203-T4-DNA-Polymerase)
25.02.13: Always check that the pipette is set to the right volume before using it.
28.02.13: When picking colonies, sterile pipette tips is an alternative to toothpicks/inoculating needle.
28.02.13: When spreading plates/inoculating cultures, inoculate the positive control LAST, in order to reduce the danger of contaminating the other samples and getting false positives.
28.02.13: After pouring, agarose gel cools down faster if there is buffer in the tray.
28.02.13: After PCR, FIRST purify the product, THEN run on gel, in order to confirm that the desired product is present after purification. (As some non-product DNA may be present also after purification, so DNA concentration is in itself not a good indicator of the amount of product present, if any.). See notes on PCR purification kit test 28.02.
Today's lesson: DON'T TRUST THE KITS, VERIFY with gel.
03.03: Calf Intestine Phosphatase (CIP) can't be heat-inactivated. After CIP treatment, restriction digests should therefore be purified (For example with a PCR purification kit).
03.03: Two restriction-digested backbones could hypothetically ligate together during the ligation step of cloning.
08.03: The sample savings in making 20 uL PCR reaction mixes instead of 50 uL are outweighed by the hassles and problems of handling sub-uL volumes. In the end, more materials might end up being used because of such problems. Stick to 50 uL reaction mixes, and making a mastermix when processing several (4+) samples.
08.03: An Eppendorf Research pipette set to 0.4 uL (rated range is 0.5-10 uL) will suck about double (!) the amount that an Eppendorf Research pipette to the same volume will.
08.03: Ergo...A microlitre is not a microlitre! It depends on who wrote the protocol and what pipette he/she used. Be consistent in your use of pipette for the same operation.
10.03: When opening a growth medium bottle, add a "notch mark" with a felt tip marker to the side of the bottle. That way, the average number of times a bottle can be opened before contamination occurs can be estimated by noting the number of marks every time a bottle is observed to be contaminated.
13.03: Plasmids MUST be linearized before analyzing size on agarose gel.
13.03: When running a gel, make sure to pour enough buffer over the gel. If the amount of buffer is too low, too high current will run through the gel. 80 V 45 min should be sufficient as a general guideline.
13.03: When evaporating water to up-concentrate a DNA sample, do not use temperature above 60-65 degrees. Yes, it will take time.
19.03: When plating out, use absolute ethanol for sterilization. 70 % ethanol burns poorly.
12.04: If an enzyme tube looks empty: Spin it down! > 20 uL can easily be stuck on the walls.
12.04: Keep all tubes and vials upright to avoid liquid coming into contact with the lid/bottleneck, which may be contaminated.
12.04: If space is available, store assumed sterile medium at 37 C to quickly uncover contamination.
When weighing out compounds (e.g antibiotics), use a wide-necked tube (ex 50 mL tube) to avoid spilling powder.
When picking colonies, consider the expected phenotype of the desired clone. If the desired clone expresses a protein from a strong promoter and the background does not, metabolic load may cause the desired clones to grow slower. Unless kept in mind, selection bias may favour larger colonies (easier to pick), and thus clones with a lower metabolic load (Might give higher rate of failures if trying to select a clone expressing a protein).
When performing miniprep, After pelletting and resuspension, cell suspension can be frozen and stored, to continue the miniprep later: http://bioinfoweb.com/Protocol-plasmid-DNA-isolation-by-alkaline-lysis-method-miniprep.htm
When incubating liquid cultures in selective media, always include a non-resistant strain as negative control, to ensure that the antibiotic is working as expected.
When transforming, always include a negative control strain to check that the antibiotics on the plate is working and that only transformed cells grow.
Using bunsen burner + ethanol-dipped glass spreader in sterile hood.
Standard operating procedures
Culturing for miniprep:
Add 25 mL LB in a 50 mL tube. Add antibiotic. Transfer 12 mL to each of two 100 mL Erlenmeyer bottles. This will give enough culture for miniprep from 2 x 10 mL or 4 x 5 mL while leaving enough to make glycerol stock.