Haynes Lab:Notebook/CRISPR Editing/2014/08/07

Project name

Main project page Previous entry      Next entry

08/07/2014 CRISPR on chromatin: transfections of g21, g22, g27 into Gal4EED/luc cells Luc-mCh swap: building the donor sequence SURVEYOR assay - C-on-C and mCherry CRISPR on chromatin: transfections gRNA21/Cas9, dox+ gRNA22/Cas9, dox+ gRNA27/Cas9, dox+ gRNA21/Cas9, dox- gRNA22/Cas9, dox- gRNA27/Cas9, dox- On 8/05/14, cells were grown in 4 mL P/S-free medium to ~80% confluence in a 6-well plate, 3 with 1 μg/mL dox, 3 without dox Luc-mCh swap: building the donor sequence Designed nested PCR primers to gradually build homology arms onto mCherry. The resulting donor should... Place mCherry in-frame upstream of the luciferase gene Add an ATG start codon onto mCherry (we had to use KAH154 as a template, where mCherry has no start) Add a double stop (TAATAG) onto the end of mCherry Add 130 bp of homology with TK-luciferase to each end SURVEYOR assay Assistance from Week 2 Team 2 Planning Target Genomic DNA (template) Primers Amplicon (bp) Expected S-Cut fragments mCherry gRNA1 CRISPR 1d qPCR 1F / P3R 329 ~229, 100 mCherry gRNA2 CRISPR 2d " 329 ~52, 277 mCherry gRNA4 CRISPR 4d " 329 ~180, 149 mCherry mock " 329 no cutting luciferase gRNA20 g20 +dox gRNA 021 US P F / gRNA 027 ES P R 1295 ~165, 1130 luciferase gRNA20 g20 -dox " 1295 ~165, 1130 luciferase gRNA29 g29 +dox " 1295 ~395, 900 luciferase gRNA29 g29 -dox " 1295 ~395, 900 luciferase mock +dox " 1295 no cutting luciferase mock -dox " 1295 no cutting mCherry annotated map at https://benchling.com/cshlsynbio/mammalian-crispr/Rc6-kah154/edit Luciferase annotated map at https://benchling.com/rene/2014-cshl-synbio-mammalian-parts/FzI-puas-tk-luc-crispr-v-chromatin/edit PCR reaction Reactions: 20 ng template DNA, 1x HF Phusion buffer, 0.2 mM dNTPs, 2.5 μL of 10 μM for each primer, 0.5 μL Phusion polymerase; in 50 μL final volume Program 98°C/ 30 sec 30x [98°C/ 10 sec, 58°C/ 15 sec, 72°C/ 15 sec] 72°C/ 10 min 4°C hold PCR product clean-up Used QIAquick PCR Purification kit Elute with 50 μL H2O Sample OD 260 260/280 ng/uL CRISPR 1d 1.562 1.81 78.1 CRISPR 2d 0.273 1.71 14.0 CRISPR 4d 1.77 1.588 79.3 g20 +dox 1.408 1.79 70.4 g20 -dox 1.057 1.78 52.9 g29 +dox 0.987 1.77 49.4 g29 -dox 0.856 1.82 42.8 +dox 1.300 1.77 65.0 -dox 1.169 1.8 58.5 U2OS mock --- --- not done yet Mismatch annealing (continued on 08/07/14) Zhang Lab approach - do not mix with a wild type reference, since ~50% or more of the amplicons in the mutated cell sample will be wild type anyway 200 ng clean PCR, 1x HF Phusion buffer, in final volume of 20 μL CRISPR 1d CRISPR 2d CRISPR 4d g20 +dox g20 -dox g29 +dox g29 -dox +dox -dox U2OS mock Thermal cycler program 95ºC/ 10 min 95ºC -- (‐2.0 ºC/s) --> 85ºC 85ºC/ 1 min 85ºC -- (‐0.3 ºC/s) --> 75ºC 75ºC/ 1 min 75ºC -- (‐0.3 ºC/s) --> 65ºC 65ºC/ 1 min 65ºC -- (‐0.3 ºC/s) --> 55ºC 55ºC/ 1 min 55ºC -- (‐0.3 ºC/s) --> 45ºC 45ºC/ 1 min 45ºC -- (‐0.3 ºC/s) --> 35ºC 35ºC/ 1 min   35ºC -- (‐0.3 ºC/s) --> 25 ºC 25ºC/ 1 min 4 ºC/ hold ∞  SURVEYOR nuclease reaction