- 16 groups, of 6 students per group
- 2 hours 20 minutes per class meeting
LAB WRITE-UP TEMPLATE PAGES
MATERIALS & METHODS - DNA AMPLIFICATION
- Reactions (100 μL total volume) contained:
- Template DNA - Kozak (BBa_BBa_J176012) inserted in plasmid vector V0120 (BBa_J176127) via standard BioBrick ligation; ~10 ng per "positive" reaction; negative reactions contained no plasmid
- Primers - Forward primer P0001 (5'-gggttttcccagtcacgacg), Reverse primer P0002 (5'-tgtggaattgtgagcggataaca). Amplicon size = 277 bp, spanning some of the vector, the BioBrick prefix sites, Kozak, the BioBrick suffix, and more vector sequence.
- 2x GoTaq colorless PCR Master Mix - Promega M7133
- A few of the samples were checked via standard ethidium bromide-stained agarose gel electrophoresis outside of class (see ). All other samples were measured via SYBRgreen staining on an LED fluorimeter.
MATERIALS & METHODS - DNA IMAGING
- SYBR Safe dye (Invitrogen) - 10,000x stock diluted to 1x in 1x TBE
- 100 μL PCR reaction was diluted in 500 μL buffer (1x TBE) to yield a 1/6 dilution of DNA
- 80 μL diluted DNA was applied to 80 μL SYBR Green dye directly on the hydrophobic slide on the fluorimeter (DNA is diluted 1/12 at this point)
- We have written several worksheets and hand-outs for this course.
- Please contact one of the instructors if you are interested in obtaining copies.