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wiki:protocols:cloning

Protocols for genotyping

The following protocol is for doing PCR using worm lysis as a template. You may use this for genotyping purpose or for amplifying desired DNA fragments from the worm.

To prepare Lysis Buffer
Add proteinase K to 1 X PCR buffer (95 ul 1XPCR buffer + 5 ul 20mg/ml proteinase K)
Proteinase K is stored in -20C.

You may estimate how many samples you need to test and scale up or down proportionally.
Aliquote 10ul of lysis buffer into each tube of a 8-strip PCR tubes.

1: Perform plate lysis
Add 50ul of M9 to your worm plate. Tilt the plate and take out 10ul to add into the PCR tube with the lysis buffer. Repeat this step until you get the worms from all plates you want to test.
Then proceed to the lysis program. 65C for 15min, 95C for 10min. The worm lysis should ideally be used immediately, but can also be store at -20 for a few days.

2: Perform PCR (20ul reaction)
Use 2ul worm lysis as template. (the remaining worm lysis can be stored at -20 if you need to use them again)
Use 10ul of the 2X PCR master mix kit (Dreamtaq).

Sample PCR set up (you can make a master mix without the worm lysis, then add the lysis separately)

2x PCR master mix 10ul
worm lysis 2ul
F primer (10uM) 1ul
R primer (10uM) 1ul
H2O 6ul
Total 20ul

PCR program

95C 3min

95C 30sec
Tm 30sec (usually 53C for my primers)
72C 1min/kb
30 cycles

72C 5min

12C on hold

1: If you genotype a deletion mutant, you may run a gel directly. Add 5ul of 6x loading dye to your sample. Mix well and load 5ul in the well.

2: If you genotype a point mutation which requires digestion, then continue with digestion.

Digestion setup:

PCR product: 4.7ul
10X Buffer: 2ul
Enzyme: 0.3ul
H2O: 13ul

Digest for ~0.5hour at 37C.
Note: most restriction enzymes works at 37C. But some RE do require a different optimal temperature. Pay attention to the user manual

Note 2: In my hands, unpurified PCR products can be directly used for digestion. The only exception so far is BfaI.

After digestion, add 4ul of 6X loading dye to each tube. Mix well, then load 10ul into the gel.

3. In some rare cases, when there is no available RE to digest, then send for sequencing.
Note: James said that unpurified PCR product can be sequenced directly. I tried once and it worked.
Use 5ul of PCR product + 10ul of H2O + 2ul of 10uM primer.

If you would prefer to treat the PCR product before sending for sequencing, then use the following way.

Add 1 µl SAP (2 U) and 1 µl HL-ExoI (10 U) to 5 µl of a completed PCR reaction.
Incubate at 37°C for 5 min.
Heat inactivate at 80°C for 10 min.

Then add 8ul of H2O and 2ul of 10uM sequencing primer, send this for sequencing.

wiki/protocols/cloning.txt · Last modified: 2021/08/27 05:26 by jliu

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