Single worm lysis and PCR

From Wikionchus

Single worm PCRs are made by lysing worms in Single Worm Lysis buffer, diluting the lysed worm in PCR water and setting up a PCR reaction with an aliquot of this DNA. An average Pristionchus pacificus worm has around 1000 somatic cells and (if an adult hermaphrodite) around 1000-2000 cells in the germ line. If, for simplicity, we consider a worm to have 2000 diploid cells this would mean 4000 haploid genomes per worm. If we consider a haploid genome to be 150 Mb in size (which is a realistic assumption, see link on Genome Structure and Size) we can calculate the average amount of DNA per worm as follows:

1bp = 660 gram/mol

1bp therefore weighs 660/ (6.2*10^23) grams

1bp multiplied by the genome size (i.e. 150 Mb) gives the weight of one genome = (660/(6.2*10^23)) * 150 *10^6 grams

which when mulitplied with the number of haploid genomes (for instance 4000) gives us the total amount of grams DNA per worm.

It is a rather small number: 6.387096e-10 grams - this equals 0,63 ng. That is enough DNA for PCR but you should take that into account if you want to run Southern Blots or any mehod that requires a lot of DNA. You need to wash off several fully grown plates of worms to get enough DNA for that.

To run single worm PCR pick 1-10 worms into 10 µl Single Worm Lysis buffer, the composition of which is given here:

• 50 mM KCl
• 10 mM Tris-HCl pH 8.3
• 2.5 mM MgCl₂
• 0.45 % NP-40
• 0.45 % Tween-20

This is essentially the same buffer that is in use for C. elegans but we do not add gelatine. Before use add 5 µl of a 20 mg/ml Proteinase-K stock per 500 µl of Single Worm Lysis buffer. To lyse worms incubate at 65°C for 1 hour and then heat inactivate Proteinase-K by heating to 95°C for 10 minutes. You can then dilute this mixture by adding sterile water (ddH₂O) - using too concentrated single worm lysis mixtures as template can actually inhibit PCR amplification. I routinely add 40 µl of sterile water - reheat the mixture to 95°C for 10 minutes and then use 4 µl of that as template DNA for a single PCR reaction of 20-25 µl total volume.

For PCR mix:

• 4 µl single worm lysis DNA
• 5 µl 5X concentrated PCR buffer
• 4 µl dNTPs 2mM
• 0.25 µl Primer Forward 20 mM
• 0.25 µl Primer Reverse 20 mM
• 0.2 µl of Taq-Polymerase
• H₂O to 25 µl total volume

Run PCR in our PE or MJR cyclers and don´t forget to book the PCR machine in advance if the lab is busy. As a general rule sequencing reactions should not be run during the day but rather overnight. If you book a PCR machine please use it within 30 minutes of the first time point you enter - any user who is looking for a machine can use it if you´re not using it by then.

Single worm lysis and PCR of SSU works also very efficiently with the following protocols:

SW DNA Prep

• Pick single worm and transfer to 20 µl of 0.25 M NaOH
• Leave overnight @RT
• Heat to 99°C for 3 min in thermocycler
• Add:
• - 4 µl 1 N HCl

• - 10 µl 0.5 M Tris-HCl pH 8.0

• - 5 µl 2% Triton X-100
• Mix
• Heat to 99°C for 3 min
• Freeze @-20°C for ≥ 3 hrs
• Heat to 99°C for 3 min

• Use 1-2 µl for PCR

SSU PCR Mix (25 µl)

• 2.5 µl 10x PCR buffer
• 2.0 µl 2 mM dNTPs
• 1.0 µl 12,5 µM primer mix (RH5401 + RH5402)
• 1.0 µl 25 mM MgCl2
• 17.3 µl H₂O
• 0.2 µl Taq DNA Polymerase
• 1-2 µl Single Worm DNA

Cycling conditions:

• 2 min, 95°C

35-40 cycles of

• 15 sec, 95°C
• 15 sec, 50°C
• 2 min, 72°C

• 7 min 72°C
• cool down

Expected fragment size: 1.1 kb

Sequencing

Dilute PCR mix 10fold

Set up

• 2 µl 5x Sequencing buffer
• 1 µl 5 mM RH5403
• 5.7 µl H₂O
• 1 µl diluted SSU PCR mix
• 0.3 µl BDT

Run standard New-BDT cycle sequencing program in thermocycler

Links

• Back to Pristionchus pacificus Protocols page