2x Worm Freezing buffer (for 1000 mls)
5.86 g NaCl
6.8 g KH2PO4 ( monobasic)
30% volume Glycerol
5.6 mls NaOH (1N)
0.6 mls MgSO4 (1M)
Instructions: Mix NaCl, KH2PO4, glycerol, and NaOH. Take to volume with distilled water. Autoclave for 20 minutes. Allow to cool to about 60C. Add MgSO4 sterilely.
Alternatively, make up all ingredients and then sterile filter. Store at 4C in 500ml bottle.
Mix 50:50 with M9 buffer in 50ml culture flasks to use for freezing.
M9 buffer for C. elegans
3.0 g KH2PO4
6.0 g Na2HPO4
0.5 g NaCl
1.0 g NH4Cl
Bring to 1 L with H2O. Autoclave or filter sterilize the solution.
Recovery Buffer for C. elegans injection
(for 40 mls)
4 mls Salmon Sperm DNA 10mg/ml stock
8mls 20% Glucose stock
96 µl 1M KCl
528 µl 5M NaCl
120 µl 1M MgCl2
120 µl 1M CaCl2
120 µl 1M Hepes pH = 7.2 (needs to be kept in 4C)
27 mls H2O
Instructions: Aliquot into 1.5 ml eppendorf tubes (1ml/ aliquot). Store at -20C in common reagents box.
General formula:
Salmon Sperm DNA 1mg/ml
4% Glucose
2.4mM KCl
66mM NaCl
3mM MgCl2
3mM CaCl2
3mM Hepes pH 7.2
Recommendations for use:
1. Take your own aliquot and store it at -20C.
2. Heat the aliquot to 65C for 15 minutes, and re-equilibrate to room temp before use. (the aliquots do NOT start off sterile). It is also good practice to heat the aliquot to 65C every time before you use it, in case it got contaminated after your initial sterilization.
3. 25 ul of recovery buffer per injection pad should be more than sufficient.
4. Keep using your aliquot until it is finished. There is no advantage to taking a new aliquot prematurely as they aren't even sterile.
5. Let us know when we are down to 8 aliquots so there is time to make more before we run out.
Proteinase K
Aliquots
Order catalog # RPROTK-RO- 3115879001 from Sigma (100mg). “Proteinase K, recombinant, PCR Grade”
When it arrives, we keep it on the top shelf of the fridge. Best to have two of these in the fridge as backup.
Making Aliquots
wear gloves, lab coat, and eye protection
Proteinase K is toxic! do not let it come in contact with your skin in either powder or liquid form!
Add 5 mls of sterile filtered, autoclaved millipore water to vial. Replace cap and mix gently until the powder has gone into solution.
Aliquot 150 ul per 1.5 ml tube.
Label top of tube "Prot.K 20 mg/ml"
Place aliquots in Freezer 1 in the common reagents rack in the appropriate box.
Worm lysis buffer
for PCR (5 mls)
250 µl 1M KCl
250 µl 1% Gelatin *
50 µl 1 M Tris pH 8.2
22.5 µl Tween-20
16.5 µl 20mg/ml Proteinase K
12.5 µl 1M MgCl2
4400 µl ddH2O
*1% gelatin is made fresh: 100 mg gelatin in 10ml water and heat in microwave)
Make a 5 ml stock and freeze at -80°C in aliquots (≈1ml or 15 ul aliquots in PCR strips/plates). I had successfully used them after freeze thaw.
50X TAE buffer
242 g Tris free base
18.61 g Disodium EDTA
57.1 ml Glacial Acetic Acid
to 1 liters ddH2O
Add the Tris free base and EDTA to approximately 700 ml DDI H2O and stir until the Tris and EDTA are dissolved. Add the acetic acid and adjust the volume to 1 liter.
The 1x TAE solution is 40mM Tris, 20mM Acetate and 1mM EDTA and typically has a pH around 8.6 (do not adjust).
5X TBE buffer
(1000 mls)
27.5 g of boric acid (CAS# 10043-35-3)
Adjust pH to 8.3 by HCl.
TBE can be diluted to 0.5x prior to use in electrophoresis.
Peptone enriched
NGM plates
(1 L and 4 L)
NaCl……………………..……………………1.2g 4.8g
Peptone……………………………………….20g 80g
Agar…………………………………………..25g X4 100g
H2O……………………………………………to 1L to 4.0L
Autoclave and then add:
Cholesterol in ethanol………………….1ml. (5mg/ml) 4.0 ml
MgSO4………………………………………..1ml (1M) 4.0 ml
KPO4………………………………………….25 ml (1M) 100 ml
NEMATODE GROWTH MEDIA (NGM) AGAR
COMPONENTS 1L 1.2L 3L 4L
NaCl 3grs. 3.6grs. 9grs. 12grs.
Bacto-Peptone 2.5grs. 3grs. 7.5grs. 10grs.
Bacto-Agar 17grs. 20.4grs. 51grs. 68grs.
dH2O ~975mls. ~1170mls. ~2932mls. ~3900mls.
1. Mix the above components and autoclave them for 35 minutes. Use a 2 liter flask for the 1 and 1.2 liter volumes. Use a 6 liter flask for the 3 and 4 liter volumes. Also autoclave the tubing for the plate pouring machine in its metal box.
2. Allow the autoclave mixture to cool at room temperature to about 60C. Do not put a thermometer in the mixture to measure the temperature. If you can handle the flask without gloves on, the mixture should be cool enough. It is imperative that this mixture remain sterile.
3. Sterilely add the following components to the autoclaved mixture in the order indicated and mix after each addition. Use good sterile technique and be sure to flame the tops of all bottles except the cholesterol.
COMPONENT 1L 1.2L 3L 4L
Cholesterol (5mg/ml in 1ml. 1.2mls. 3mls. 4mls.
95% EtOH)
CaCl2 (1M sterile) 0.5mls. 0.6mls. 1.5mls. 2mls.
MgSO4 (1M sterile) 1ml. 1.2mls. 3mls. 4mls.
KH2PO4 (1M pH6 sterile) 25mls. 30mls. 75mls. 100mls.
4. Set up plate pouring machine as described in the Plate Pouring Protocol. In addition to the tubing, you will need to have a 400ml. beaker, a 2 liter flask, a hemostat, and paper towels.
5. Approximate plate volumes are as follows:
9cm. plates-~30mls.
6cm. plates-~10-15mls.
3cm. plates-~5mls.
microtiter plates-~2.5mls.
Gibson assembly mix
The original protocol was described in Nature Methods: http://www.nature.com/nmeth/journal/v6/n5/full/nmeth.1318.html
The mix is from Nature Protocols:
http://www.nature.com/protocolexchange/protocols/554#/procedure
Here are the components for the mix. These are typically already made and aliqouted.
Enzyme mixes:
Prepare 5X ISO buffer. Six ml of this buffer can be prepared by combining the
following:
3 ml of 1 M Tris-HCl pH 7.5
300 µl of 1 M MgCl2
600 ul of 10 mM dNTP mix
300 µl of 1 M DTT
1.5 g PEG-8000
600 µl of 50 mM NAD
Add water to 6 ml (ends up very close to 6 mL, no need to add water)
Aliquot 350 µl and store at -20 °C
Master assembly mixture: This can be prepared by combining the following (for 500 ul and 1200 ul mix respectively):
320 µl 5X ISO buffer; 100 ul 5x ISO
0.64 µl of 10 U/ µl T5 exo; 0.2 ul 10 U/ul T5 Exo
20 µl of 2 U/µl Phusion pol; 6.25 ul 2U/ul Phusion Polymerase
160 µl of 40 U/µl Taq lig ; 50 ul 40 U/ul Taq Ligase
Add 700 ul water; 218.5 ul water.
Reagent catalog numbers:
T5 Exonuclease from Epicentre. Cat # T5E4111K
NAD+ from NEB: Cat # B9007S
Taq ligase from Enzymatics: Cat# L6060L
dNTPs & Phusion from NEB
Aliquot 15 µl and store at -20 °C.
For the 500 ul mix (right) that gives 33 aliqouts of 15 ul.
For the 1200 ul mix (left) that gives 80 aliqouts of 15 ul.
This assembly mixture can be stored at -20 °C for at least one year. The enzymes remain active following at least 10 freeze-thaw cycles.
This mix is ideal for the assembly of DNA molecules with 20-150 bp overlaps. For
DNA molecules overlapping by larger than 150 bp, prepare the assembly mixture
by using 3.2 µl of 10 U/ µl T5 exo Gibson Reactions:
Add 3 ul of Gibson mix for every 1 ul of DNA.
Incubate at 50C for 1 hour.