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Protocols for worm media and maintenance

Most of the protocols can be found on wormbook. The ones below are from BioProtocols. He, F. (2011). Common Worm Media and Buffers. Bio-101: e55. DOI: 10.21769/BioProtoc.55.

Before using any of these protocols, please cross-check with other sources. recalculate the given weights with the molar weights on our chemicals. When you verified a protocol, make a note 'verified by [NAME]' under the headline.

Stock solutions

  • Cholesterol 5 mg ml-1 in 95% EtOH
  • 1 ml 1 M CaCl2
  • 1 ml 1 M MgSO4
  • 25 ml 1 M potassium phosphate (pH 6)
  • Nematode growth medium (NGM) liquid

1M potassium phosphate (pH 6)

Alternatively: Make the following two solutions separately, then mix to obtain correct pH (6.0):

A. For 250 ml 1M KH2PO4 (monobasic): Add dH2O to 34.0 g KH2PO4 until final volume (250 ml) is obtained

B. For 200 ml 1M K2HPO4: Add dH2O to 45.6 g K2HPO4 *Make sure the salt in the solutions is completely dissolved.

  1. Add K2HPO4 solution to KH2PO4 solution to bring the pH up from 4.0 to 6.0 (will take about 100 ml of K2HPO4).
  2. Autoclave 15 min. liquid cycle
  3. Store at room temperature.

Alternatively: For 1 liter, dissolve 136.1 g KH2PO4 in about 800 ml dH2O, then adjust to pH 6.0 with solid KOH (approx 15 g) before bringing up to volume. Make 100 ml aliquots and autoclave. Jun's note: you may also use ~53ml of 5M KOH solution.

Nematode growth medium (NGM) agar

For the maintenance of worms on plates. For liquid NGM just leave out the agar. For 1 liter medium

  • 3 g NaCl
  • 17 g agar
  • 2.5 g peptone
  • 1 ml cholesterol (5 mg ml-1 in 95% EtOH)
  • 975 ml H2O

Autoclave, and then add the following sterile solution (autoclaved)

  • 1 ml 1 M CaCl2
  • 1 ml 1 M MgSO4
  • 25 ml 1 M potassium phosphate (pH 6) (to avoid precipitation, mix between addition of MgSO4 and potassium phosphate

Need to add streptomycin (300 ng ml-1) if plate is used for seeding bacterial food E. coli OP50-1. Typically pour 60 x 15 mm plate and store NGM plates in plastic boxes with covers at room temperature.

Seeding NGM plates with OP50

Day 1: grow an overnight culture of OP50 in LB medium. Pick a single colony from an LB plate using a sterile tip (stored at 4C). Place tip in a sterilized Erlmeyer flask with approx. 200ml of LB broth. Shake at 37C.

Day 2: Add 100ul of the OP50 culture to NGM plates and swirl to distribute. Let it dry overnight on the bench. Plates can be stored for 3 Weeks in the cold room.

Plate size OP50 Volume
3.5 cm 34 ul
6 cm 100 ul
10 cm 275 ul

Imaging plates

These are modified versions of the standard NGM plates . For 1 liter medium

  • 3 g NaCl
  • 17 g agarose (replace agar with agarose)
  • 2.5 g peptone
  • Do not add Cholesterol. It creates background
  • 975 ml H2O

Autoclave, and then add the following sterile solution (autoclaved)

  • 1 ml 1 M CaCl2
  • 1 ml 1 M MgSO4
  • 25 ml 1 M potassium phosphate (pH 6) (to avoid precipitation, mix between addition of MgSO4 and potassium phosphate

S-basal medium

For liquid culture of worms and use in microfluidics.

For 1 liter medium

  • 5.8 g NaCl
  • 50 ml 1 M potassium phosphate (pH 6)
  • 1 ml cholesterol (5 mg ml-1 in 95% EtOH)
  • 950 ml dH2O

Autoclave, and then add the following sterile solution (autoclaved)

  • 3 ml 1 M CaCl2
  • 3 ml 1 M MgSO4
  • 10 ml trace metals solution
  • 10 ml 1 M potassium citrate (pH 6.0)
  • 10 ml 100x Nystatin (antifungal agent, keep in freezer; we rarely add this).

500 ml trace metals solution

  • 0.346 g FeSO4.7H2O
  • 0.930 g Na2EDTA
  • 0.098 g MnCl2.4H2O
  • 0.144 g ZnSO4.7H2O
  • 0.012 g CuSO4.5H2O

Sterilize by autoclaving. Keep in dark (wrap in foil).

100 ml of 1 M potassium citrate

dissolve 21.02 g citric acid, monohydrate in 80 ml and adjust to pH 6.0 with solid KOH (approx 17g) before bringing up to volume.

Worm M9 buffer

  • 3 g KH2PO4
  • 6 g Na2HPO4
  • 5 g NaCl

Add H2O to 1 liter. Sterilize by autoclaving. After solution cools down, add

  • 1 ml autoclaved/sterile 1 M MgSO4.

Jun's note: you can also make 10X M9 solution by X10 of each components.

Worm lysis solution

This makes 100 ml 2x worm lysis solution. It is used for worm egg synchronization.

  • 50 ml ddH2O
  • 10 ml 10 M NaOH
  • 40 ml Clorox bleach

Make fresh and store at 4 °C up to one week.

Spot bleach protocol by Jun This protocol is to be used if you want to de-contaminate your worms when there are fungi or bacteria contaminations on the plate.
1) Add ~50ul of bleach solution to a seeded NGM plate. Avoid adding to the bacterial lawn.
2) Pick >5 gravid adults from contaminated plates and add the worms to the bleach solution on the plate.
3) Use the worm pick to press the worms a bit to help seperate the worms. Alternatively, you can also shake the plate gentally to help break-off.
4) leave overnight and the hatched L1 will crawl out.
5) Optional: transfer at least 4 L1s to a new plate just in case there are fungi survived (which occasionally happen when the original plates are heavily contaminated.

Canonical bleach protocol by Jun This protocol is to be used if you want to collect many synchronized L1s or to de-contaminate your worms when there are fungi or bacteria contaminations on the plate (this will give you many more embryos than the spot bleach).
Note: For behavioral assays, it is NOT recommended to use bleaching to obtain synchronous worms. Use egg laying method instead.\\. 1) Wash off 1 plate with many gravid worms with 1ml of M9.
2) Add them to a 1.5ml of Eppendorf tubes.

  • . Label the tubes well. The label might come off during the washes in the following steps.

3) Spin down at 1600g for 30~60sec.
4) Aspirate the supernatant and wash with 1ml of M9.

  • . It is ok to leave 50~100ul of liquid at the bottom. Otherwise you risk of losing worms.

5) Repeat step 4 for 2~3 times (until supernatant is clear).
6) Aspirate and add 1ml of bleaching solution.
7) Vortex or shaking in hand for 2min.
8) Spin down at 1600g for 1min.
9) Aspirate and add 1ml of bleaching solution.
10) Vortex or shaking in hand until you can see embryos under the microscope and that the worms are completely broken.

  • . No longer than 2min, otherwise you will kill the embryos.
  • . If you are just doing this for de-contamination, it is ok to have unbroken worms. If you are doing it for synchronization, note that the broken worms can be food for newly hatched L1 worms, thereby causing asynchronous population.

11) Spin down at 1600g for 1min.
12) Wash with 1ml of M9 at least three times.
13) Add 1ml of M9 and rotate at room temperature overnight.

  • . If you do not need to synchronize worms, you may just add the embryos from step 12 onto a new plate.

Bleaching solution (recipe for 100ml) by Jun

30 ml	Sodium hypochloride
15 ml	5M KOH 
55 ml	ddH2O

It always works the best when made fresh. However, it can be stored at 4⁰C for a week.

Freezing C. elegans using Liquid Freezing Solution

(from wormbook)

Equipment and Reagents

  • S Buffer [129 ml 0.05 M K2HPO4, 871 ml 0.05 M KH2PO4, 5.85 g NaCl]
  • S Buffer (see above) + 30% glycerin (v/v) (autoclave)
  • 1.8 ml cryotube vials
  1. Use one large, 2-3 medium, or 5-6 small NGM plates that have lots of freshly starved L1-L2 animals. Wash the plates with 0.6 ml S Buffer for each vial you will freeze. Collect liquid in a sterile test tube.
  2. Add an equal volume of S Buffer + 30% glycerin. Mix well.
  3. Aliquot 1.0 ml of mixture into 1.8 ml cryovials labelled with strain name and date.
  4. Pack the cryovials in a small styrofoam box with slots for holding microtubes or use a commercial styrofoam shipping box.
  5. Place the box in a −80°C freezer overnight (or for at least 12 hours).
  6. The next day transfer the vials to their permanent freezer locations. Thaw one vial as a tester to check how well the worms survived the freezing

Freezing C. elegans using Soft Agar Freezing Solution

Equipment and Reagents

  • Soft Agar Freezing Solution [0.58 g NaCl, 0.68 g KH2PO4, 30 g glycerol, 0.56 ml 1 M NaOH, 0.4 g agar, H2O to 100 ml (autoclave)]
  • 1.8 ml cryotube vials

Methods

  1. Melt Soft Agar Freezing Solution in autoclave or microwave and place in 50°C water bath for at least 15 minutes.
  2. Use one large, 2-3 medium, or 5-6 small NGM plates that have lots of freshly starved L1-L2 animals. Wash the plates with 0.6 ml S Buffer for each vial you will freeze. Collect liquid in a covered sterile test tube and place in ice for 15 minutes.
  3. Add an equal volume of Soft Agar Freezing Solution to the test tube. Mix well.
  4. Aliquot 1 ml of mixture into 1.8 ml cryovials labelled with strain name and date.
  5. Pack the cryovials in a small styrofoam box with slots for holding microtubes or use a commercial styrofoam shipping box.
  6. Place the box in a −80°C freezer overnight (or for at least 12 hours).
  7. The next day transfer the vials to their permanent freezer locations. Take a scoop of frozen mixture from one vial as a tester to check how well the worms survived the freezing

C. elegans growth speed

How to stage C. elegans

Determination of larval and adult stages (with Ingo) [Probably written by Florian Aeschimann]

Hallmarks of the different larval and adult stages of C.elegans visible with DIC under the Zeiss microscope (63x objective).

General remarks: Worms on the plates most of the time lie on their sides, the gonads/vulva are thus seen on one side (ventral), whereas the alae are seen in the middle of the worm surface (lateral).

Annuli are visible in all larval and adult stages.

Alae occur only on the cuticula of the L1 larvae and the adult worms (although the alae of the adult cuticula are already visible in late L4).

L1 Small gonad with only few gonad cells (only Z1-Z4 (picture C) in early L1, with their descendants in late L1) Alae on the worm surface

L2 Small gonad with an increased number of germ cells (picture D) No alae on the worm surface

L3 Larger gonad (extended gonad arms) with many germ cells (picture F) No vulva

Early L4 Gonad arms are reorientated (visible turn/bending is lab definition for early L4, otherwise still L3) Vulva is seen as a very small invagination

Mid L4 Gonad arms turn back about halfway Vulva is seen with a Christmas tree structure (picture G)

Late L4 Gonad arms turn back all the way Vulva is seen with a Christmas tree structure or further developed, but with a cuticula covering the vulva Alae structures are already visible from the adult cuticula below the L4 cuticula

Young adult Gonad arms turn back all the way and overlay each other Vulva is developed, without a covering cuticula Alae on worm surface No embryos visible

Adult Gonad arms turn back all the way and overlay each other Vulva is open to the outside (picture H) Alae on worm surface Embryos visible (about 4 hours after L4-adult molt?)

To precisely stage L3 and L4 worms, see below for vulva morphology comparison. Please note the timing in the following picture refers to the growth rate at 22 degrees. PMID: 24945623

Protocols for Using Freeze-dried OP50




General practice for microbiology

Bullet point version

1) When labeling your plates, always label at the back (write the date and strain name)
2) When you put the plates in the incubator or fridge, always invert the plate (bottom up)
3) When you inoculate into growth media (such as LB), always pick a single colony from a plate (rather than scratch from a bacterial lawn)
4) Do not grow the liquid culture of bacteria longer than 16 hours at 37 degree with shaking (this also applies when you grow bacteria on plates, but could be slightly longer)
5) Do not inoculate from bacteria (such as OP50) that have been kept in the fridge for longer than a month. (I will prepare a fresh OP50 plate at the beginning of every month)
6) If you work with OP50-dsRed, use bacteria plate as fresh as possible (ideally no longer than 1~2 weeks), otherwise you get worse expression of dsRed protein.
7) It is not recommended to directly inoculate from -80 stock
8) When you need to use a strain from -80 stock, steak single colonies first, then inoculate from the single colony (you can find training videos of streaking single colonies and inoculation from Dropbox (Scholz Lab)\Scholz Lab's shared workspace\Training Resources\Videos )
9) For liquid culture (such as OP50 inoculated in LB media, which we use for seeding worm plates), it can be kept in the fridge for no more than 2 weeks. Mix well before you use it again.
10) When you grow a large bottle of OP50 and want to inoculate, it is recommended to alioquot first and then use the aliquot to seed to avoid contaminating the stock.

Why version
1) When labeling your plates, always label at the back (write the date and strain name)

  a.	If you label only on the lids and work with multiple plates, you might mix up the lids

2) When you put the plates in the incubator or fridge, always invert the plate (bottom up)

  a.	Condensed water will accumulate on the lid during incubation/storage. 
  b.  When the water drops, it will mix up colonies

3) When you inoculate into growth media (such as LB), always pick a single colony from a plate (rather than scratch from a bacterial lawn)

  a.	Colonies may differ from one to another, this is especially true when you do clonings
  b.	Spontaneous mutations may arise and thus colonies may differ  

4) Do not grow the liquid culture of bacteria longer than 16 hours at 37 degree with shaking (this also applies when you grow bacteria on plates, but could be slightly longer)

  a.	When you grow the liquid culture for too long, the bacteria will over grow and become bad quality (in a simplified point of view)
  b.	If you use media with antibiotics, the antibiotics will be degraded over time and you will end up with growing bacteria that lose the plasmids.

5) Do not inoculate from bacteria (such as OP50) that have been kept in the fridge for longer than a month. (I will prepare a fresh OP50 plate at the beginning of every month)

  a.	Spontaneous mutations may arise and there will be random recombination in the bacteria genome.

6) If you work with OP50-dsRed, use bacteria plate as fresh as possible (ideally no longer than 1~2 weeks), otherwise you get worse expression of dsRed protein.

  a.	Treat it like protein expression of dsRed during bacteria culture. For optimal bacterial protein expression, fresh colonies will give better yield.

7) It is not recommended to directly inoculate from -80 stock

  a.	Spontaneous mutations may arise and you may inoculate from a mixture of bacteria with differently mutated background.

8) When you need to use a strain from -80 stock, steak single colonies first, then inoculate from the single colony (you can find training videos of streaking single colonies and inoculation from Dropbox (Scholz Lab)\Scholz Lab's shared workspace\Training Resources\Videos )
9) For liquid culture (such as OP50 inoculated in LB media, which we use for seeding worm plates), it can be kept in the fridge for no more than 2 weeks. Mix well before you use it again.

  a.	The bacteria will precipitate at the bottom. 

10) When you grow a large bottle of OP50 and want to inoculate, it is recommended to alioquot first and then use the aliquot to seed to avoid contaminating the stock.

Practical guide to C. elegans genetics

I am writing this as a practical guide for you when you read worm papers and/or design your experiments. As a practical guide, I only write the most commonly used terms and examples. If during your reading or experimentation, you find something cannot be explained by the following terms, then feel free to discuss with me and I will explain the rare cases with you.

Part 1: Different alleles

1) Null allele:
Definition: “A null allele is a nonfunctional allele caused by a genetic mutation. Such mutations can cause a complete lack of production of the associated gene product.”
Causes: Commonly caused by a deletion in the gene (most common), or mutation that abolish a splicing site (common), or mutation in the start codon (least common).
Convention: Some papers use (0) to indicate an allele is a null, eg: mec-10(0). Note that (0) is not an allele name. It is just for simplicity to show that this particular allele is null.
Heterozygous phenotypes: a heterozygous will usually show wild-type phenotype. Exception: in some cases, when one copy of the wild-type allele is not sufficient to make functional product, then the heterozygote will show some defect. This is called “haploinsufficiency”. A hypothetical example is that gene R makes red pigment of a flower, and r is a null allele. RR will produce red flower, rr will produce white, and Rr will produce pink flower if it is haploinsufficient. When one copy of wild-type allele is sufficient, Rr will show red flower and this is more common than haploinsufficiency.

2) loss-of-function allele
Definition: Loss-of-function allele results in the gene product having no function (being wholly inactivated). When the allele has a complete loss of function, it is the same as null allele.
Convention: Some papers use (lof) to indicate an allele is loss-of-function, eg: mec-10(lof).
Heterozygous phenotypes: the principle is the same as null.
Note: on Wikipedia, they group together the wholly loss-of-function and partial loss-of-function. For easier understanding, I separate them here.

3) partial loss-of-function allele (reduction-of-function)
Definition: partial loss-of-function allele results in the gene product having reduced function (being partially inactivated).
Convention: Some papers use (rof) to indicate an allele is reduction-of-function, but this is not common.
Heterozygous phenotypes: the principle is the same as null.

4) gain-of-function allele
Definition: Gain-of-function allele changes the gene product such that its effect gets stronger (enhanced activation). For example: a protein only becomes active when there is a trigger (eg: insulin activates the insulin receptor when you blood level goes up, then the insulin receptor becomes active). A mutation that will make the protein constitutive active is called gain-of-function.
Convention: Some papers use (gof) to indicate an allele is gain-of-function.
Heterozygous phenotypes: it will show the gof phenotype. A hypothetical example is that gene R, which produces red pigment, is only active when there is sunlight. Then the flower is usually white and it turns red when there is sunshine. If there is a gof allele called r, which is active no matter there is light or not (called constitutive active), then the Rr will produce red flower.

5) dominant negative allele
Definition: dominant negative allele makes an altered gene product that acts antagonistically to the wild-type allele.
Convention: Some papers use (DN) to indicate an allele is dominant negative. But this is not as common.
Heterozygous phenotypes: it is usually complicated and needs to be studied case by case. Sometimes it shows a defect similar to a null, as the DN allele will mask the wildtype allele and behave as if there is no wt product (therefore null). A simplified analogy is a broken vending machine that can take coins but won’t give you anything (the DN allele). When you switch to a functional vending machine (another wildtype allele), you find out that you don’t have any coins left. This is how the DN allele would mask the wt allele.
Sometimes dominant allele will give a defect even worse than a null. I don’t want to make things too complicated so I am not stating any examples or analogies here. Of course, you can talk to me if you want to know more.

6) temperature sensitive allele
Definition: Temperature-sensitive mutations are mutations that exhibit a mutant phenotype at high or low temperatures (called restrictive temperature or non-permissive temperature) and a wild-type phenotype at normal temperature (called permissive temperature). Temperature-sensitive mutants are valuable tools for geneticists, particularly in the study of essential genes.
Convention: most cases abbreviated as ts.
Note: be careful about the interpretation of the mechanism of the ts allele. Sometimes, it is just a guess.

Part 2: Common mutation types

1) point mutations
Eg: G gets mutated to A; and C to T. Note: these are the two most common types of point mutations in C. elegans after EMS mutagenesis. It is more difficult to genotype point mutation than deletion/insertion.

A point mutation usually only changes one amino acid in the protein. However, it can still create null mutations, when i) it creates an early stop codon (eg: CAA to TAA); ii) it abolishes the start codon; iii) it changes a splicing site.

Three types of point mutations:
1: nonsense mutation: a mutation that results in stop codon.
2: missense mutation: a mutation that changes the amino acid.
3: silent mutation (same sense mutation): a mutation that does NOT change the amino acid. For example, when CCA is mutated to CCT, both of them encode Proline, therefore it is called silent mutation. Most of the cases (with few exceptions), a silent mutation will not result in defect.

2) deletion and insertion

Eg: a certain length of DNA fragment (from one base pair to a few kilobases) gets deleted or inserted in chromosome. When there is a large deletion or insertion, it likely creates null allele.

3) other types
Other types of mutations, such as inversion, duplication, are much less frequent and not explained here.

Part 3: what allele to choose, when you design your experiments?

Here comes the most practical part, when you design your experiments, what allele should you choose?

General guideline: look for null alleles as the start. If both null alleles and a point-mutation give similar defect of interest, always go for the null allele, because it is much easier to interpret the mechanism when one uses the null allele.

In some cases when a point-mutation gives a stronger defect of interest than the null mutant, or when a null mutant is not available, then go for the allele which gives the strongest phenotype related to your project.

Part 4: a few WHYs
Here comes the less practical part, about WHYs.

Q: Why is it better to work with null mutants?
A: It is easier to interpret the result when the protein product is not present at all. As for point mutation, the interpretation of the mechanism might prove to be wrong some years after the publication.

Q: Point mutation can also result in a null mutation. What is the difference to the null mutation created by deletion?
A: when a point mutation creates an early stop codon, or abolishes the start codon or the splice site, this gene can no longer make a functional product and therefore behaves similar to a null. However, due to alternative splicing, sometimes a point mutation only abolish one or more isoforms and therefore it is not a complete null. Additionally, the isoforms of some genes use different start codons, and therefore mutating one start codon only affects one isoform. Please keep in mind that some of the isoforms or different start codons are discovered many years after these genes have been extensively characterized.

One example is that a very hot gene, PTEN, was discovered in 1997. However, in 2013, another start codon was discovered and results in a longer PTEN (named as PTEN-L). The community has to publish a new paper to revise the amino acid numbering: “A unified nomenclature and amino acid numbering for human PTEN”. The publications on PTEN is probably more than that of any worm genes. Yet, there are surprising findings of a longer PTEN that has been ignored in the field for almost 20 years.

Therefore, it is possible that a null allele may not be a true null. I don’t mean to scare you from using point mutations. But you should keep this in mind when you get some unexpected findings.

Below is an example of how mutations in one splice site will not affect the other isoform. The mutation (*) will only affect isoform A but not B. Imagine that if isoform B had not been discovered at the time people published their paper, then they would have thought this mutation creates a null allele.

Below shows the alternative splicing in wildtype situation.

Below shows how a point mutation would affect one of the isoforms due to wrong alternative splicing.

Below I made an example that shows how a nonsense mutation would affect the product.

Q: How to interpret a reduction of function from a point mutation
A: you should read the publication(s) carefully to see whether their model of mechanism is solid. Sometimes the authors suggested a model that was not very well supported by their data.

Q: why do you need to backcross a mutant strain from CGC?
A: many C. elegans mutant strains are isolated from mutagenesis screen. This will create many background mutations. Backcrossing will help to “clean up” the background mutations. Usually one needs to backcross it to N2 for ~6 rounds before using the mutant. See below for representative picture about how backcrossing reduces background mutations. After each round of BC, the background mutations get less.
If one does not backcross and uses the mutant directly and observe a defect, this defect may be due to a background mutation rather than the mutant of study.
To be more strigent, some people like to use two different alleles of the same gene to prove that both of alleles give similar results. This will also help to rule out the possibility of background mutation.

wiki/protocols.txt · Last modified: 2020/09/24 00:02 by jliu

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