Pcr

One way to make transgenic animals in C. elegans we use a microinjection technique. Briefly, a DNA construct (plasmid, cosmid or YAC) or PCR product with your gene(s) of interest is mixed with a co-injection marker and injected into the distal gonad (syncytium). The injected DNA is taken up into the mature oocyte's nucleus. The DNA exists as an extrachromosomal array (i.e. not integrated in the chromosome) which segregates randomly and can be lost, that is why we need a marker to follow which animals have the array. F1 progeny that show the co-injection marker phenotype are picked and transgenic lines are established by keeping those animals that segregate the array in their F2 generation. Usually 1 in 10 F1 progeny with the array will give you a transgenic line.

DNA Extraction via Chelex

The purpose of this procedure is used to chew up excess primers and remove excess dNTPs from your PCR product. This proceedure is necessary to ensure clean and readable DNA sequences. Work in HIGH DNA part of lab.

Basic proteinase K treatment

One of the fastest way to screen bacterial colonies. With a PCR machine that takes 24 tubes you can routinely screen 22 colonies + 1 negative + 1 positive control.

PCR mutagenesis is a method for generating site-directed mutagenesis. This method can generate mutations (base substitutions, insertions, and deletions) from double-stranded plasmid without the need for subcloning into M13-based bacteriophage vectors and for ssDNA rescue. This method uses a proof-reading polymerase to read all the way around a plasmid and thus incorporate the primer as the new (mutant) sequence. Only a few (say 12) PCR cycles are performed on a relatively large amount of plasmid template to minimise the chance of expanding PCR sequence errors.

A method for direct cloning a PCR product, by the T-vector technique. This is cheap and easy way to clone PCR products with A 3’ overhangs.

5X Long PCR Buffer (store in 1ml aliquots in -20C).

Single Worm PCR for use with C. elegans