Haynes Lab:Notebook/CRISPR Editing/2016/09/03

From OpenWetWare
Jump to navigationJump to search
Owwnotebook icon.png Project name Report.pngMain project page
Resultset previous.pngPrevious entry      


ChIP incubation ran 12 hours overnight.
Added washed beads to IgG samples
Washed FLAG beads 4x with 1mL TBS buffer, 10 min nutation between washes
after last wash, removed TBS, quick spin, remove remaining TBS with P20
added 100ul elutiion buffer to each sample
Added 50ul elution buffer to inputs
put at 65deg for 8 hours

Washed IgG bead samples after 3 hour nutation at 4deg, 6x with RIPA buffer, 10min nutation between each wash
washed twice with TE pH 7.6, 3 minute nutation between washes
removed TE after last wash, quick spin, removed remaining TE buffer with P20
added 100ul elution buffer to each sample
put at 65deg for 5 hours

30 mins at 37deg with 1ul RNase A
2 hours at 62deg with 0.5ul proteinase K

purify with Sigma columns

Started some cultures from the g032 plate

4 1mL cultures for 3 hours
2mL culture -> 100mL media
grew for 5 hours
maxi prep all 100mL