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tales from the ols building Episode 1: Cloning

2/27/2019

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My cloning worked!!! 
It took a good 6 months, then briefly on and off for a year and a half, but I'm happy to report that apparently persistence works even when you've completely given up all hope!
If you're like me with your cloning attempts, you've gotten great amplification with your insert, you've already got the vector ready to go, AND you've probably even successfully cut your vector (AND insert!) with several restriction enzymes AND THE BANDS look great, HOWEVER..dun dun dun!!! Then comes the dreadful part. The gel extraction!
Aghh I kept loosing everything here. Or even at the PCR cleanup step. Was it the water? The pH of the buffer? Old EtOH? Come on internet, someone has to have a solution to my cloning woes!!! So many questions, so little time, and nothing's working!!! UNTIL... I had tried so many times and enough time had passed for me to really give up on the project completely that when I thought about doing this again I had a renewed sense of what the heck was going on here.

1. DO NOT LISTEN TO NEB!!!
Ok, NEB has some great tools and advice AND they are actually awesome, however, they recommend completing a digest in a 50uL reaction volume. Doing this was the absolute worst. Especially if you're already starting with a small amount of DNA. It only makes sense that you would want to keep the molecules as tightly packed/close together as possible to increase the chances these things will run into each other. This may sound like common sense to a veteran clone-master, but to this gumby I was just following the instructions!  This was my main problem.
SOLUTION: KEEP ALL YOUR REACTION VOLUMES (FOR PCR PURIFICATION, GEL EXTRACTION, & LIGATION) ANYWHERE BETWEEN 10-20uL. If you can't do this, then your starting material is already too diluted & you NEED MORE COWBELL! This brings us to #2:   

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