swPCR of SPD-2 worms
After a few days of injecting the worms, and progeny was picked (by my supervisor), it was time to screen the progeny to see whether any of them had taken up the insert. This was done by lysing the progeny, then amplifying the inserts, before running a gel to see if the bands for the endogenous and exogenous SPD-2 is present.
Over the weekend, my supervisor picked 100 progeny of the injected SPD-2 worms, and although 100 worms may seem like a lot, it is likely for only 1 of those worms to have the insert - so the more worms, the more likely we will have a positive result! From these 100 worms, I picked and placed them individually into 20ul of 1x PCR buffer before freezing them at -80C. This step helps to ensure that the insert will be taken up by the worms. After freezing, I then lysed these worms to release their DNA so they can be screened. This was done using proteinase K, and was heated in the PCR machine for 1 hr. I then froze the worms again in the -80C freezer before finally preparing a 1.3% agarose gel to screen for the insert.
These steps weren't difficult as I was following a standard protocol, however, picking, pipetting and repipetting 100 worms was the challenge! As 100 worms are a large amount, the work load had to be split into two days, allowing me to focus on the first 50 worms on one day, then the next 50 worms the next day.
Results from the swPCR have shown a few worms with the insert! As the primers would bind to the endogenous and exogenous SPD-2, we were hoping to see at least one worm with two bands. Luckily, atleast two worms appeared to have the insert, with some other worms showing a faint second band - indicating a potential succesful insertion. This step was most exciting, as we could finally see all parts of the project coming together and actually being present within the worms!
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