Haynes Lab:Notebook/CRISPR Editing/2016/02/29

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02/29/2015

Plated Luc14s in 12-well plate, 0.1x10^6 cells/well, for transfections tomorrow

Ligation for pX330_dCas9_EGFP
Backbone calculations for 50ng

' conc (ng/ul) length (bp ul for 50ng
pX330A_dCas9_1x4 52 8939 0.96

insert calculations for 1:2 ratio

' conc (ng/ul) length (bp ul for 2:! ratio
gB009 1.60 838.00 5.86
' ul
backbone 0.96
insert or water 5.86
NEB 10x ligation buffer 2
NEB T4 ligase 1
water 10.18
total 20

Mixed gently, inc at RT for 30 mins
Add 50ul cells to each reaction
inc on ice for 30min, heat shock at 42 for 35s, ice for 2 mins, shake in 750ul SOC for 1 hour, plate



Ligation for MV14
Backbone

' conc (ng/ul) length (bp ul for 50ng
MV13-KAH60 48 6240 1.04

Insert

' conc (ng/ul) length (bp ul for 2:! ratio ul 1:100 dilution
gB014 64 122 0.03 3.05
' ul
backbone 1.04
insert or water 3.05
NEB 10x ligation buffer 2
NEB T4 ligase 1
water 12.9
total 20

Mixed gently, inc at RT for 30 mins
Add 50ul cells to each reaction
inc on ice for 30min, heat shock at 42 for 35s, ice for 2 mins, shake in 750ul SOC for 1 hour, plate

Running gel for western
Got NuPAGE LDS Sample buffer 4x out of 4deg fridge
Got Invitrogen BenchMark Prestained Protein Ladder from -20degC protein reagents box
Got 1M DTT out of -20degC freezer
Got my protein samples out (Luc14 cell lysates prepped 2/19/16)
Set heat block to 100degC

Gel lanes
stock: ladder
sample prep: Luc14 1 (14 uL protein)
sample prep: Luc14 2 (14 uL protein)
sample prep: dummy (14 uL water)

  1. ladder
  2. Luc14 1
  3. Luc14 2
  4. dummy
  5. ladder
  6. Luc14 1
  7. Luc14 2
  8. dummy
  9. ladder
  10. Luc14 1
  11. Luc14 2
  12. dummy


Ladder is prepped and ready to go.
Need to make sample buffer mix for the other 9 lanes (protein samples and dummy lanes).

Mastermix for sample buffer
Mix MM, add 6ul to each sample/

' 1 lane 9 lanes (x10)
4x Loading Buffer 5 50
1M DTT 1 10
Protein/water 14

heat at 100degC for 5 mins, cool in rack at RT

Prep gel
Gel/ Buffer

NuPAGE 4-12% BisTris
NuPAGE® MOPS SDS Running Buffer (20%MOPS diluted in DI water from sink)

remove the strip from the gel, put premade gel into box with the open part facing the outer chamber, fill inner chamber to cover the comb, outer chamber to cover the bottom of the gel.
Add 500ul antioxidant to the inner chamber
remove the comb



load samples and ladder (5ul ladder)

ladder in lanes 1 and 9 might have a bit less than 5ul

run gel for 1 hour at 120V (until lowest dye band ran just past the third ridge in gel box)


Transfer and blocking following this protocol.
Remove the gel from the box, rinse with DI water, scrape the exposed gel slit on the bottom to get all the gel from that spot. Rinse the gel gunk away. Crack the plastic apart but don't pull it apart until the plastic is unstuck. With the gel submerged in a tupperware filled with DI water, separate the plastic pieces. Pay attention to which plastic part the gel is sticking to. Pay attention to the direction of your gel. Get the gel floating in the DI water.
Transfer the gel to nitrocellulose paper using the transblot machine. Make sure to keep the orientation.

list
biorad
1 minigel
mixed mw
run
A-run


Should have no bands in the gel and bands on the nicrocellulose. Rinse the nitrocellulose in DI water. Move the nitrocellulose to a smaller dish. Pour Ponceau stain over to just cover it. Orbital shake gently for 5-10 minutes. Rinse the Ponceau in the sink in DI water until the background is white.
Image the nitrocellulose on the syngene PXi machine. Remove the UV box and replace with the black background piece. Blots>visible blot>Ponceau red> -> or used saved protocols "ponceau stain". Keep nitrocellulose in water until time to image. Keep water nearby so you can quickly transfer. Need to make sure it doesn't dry out. Take an image.

Screenshot 2016-03-12 20.40.35.png

Cut the nitrocellulose to separate the pieces that will be bound with different antibodies. add notches to one corner so you remember the orientation
To block the nitrocellulose, move to a new dish and pour 5% BSA, 1% tween, 1% PBS over. Ok to stack the nitrocellulose. Keep still at RT for one hour (went about 1.5hours)

Western primary stain
Make or thaw more blocking buffer (5% BSA, 1%PBS, 1% tween, bring to volume with DI water from tap).
Make parafilm pouches! Cut a strip of parafilm that can be folded over to make a pouch just a few millimeters wider than each membrane. Lay the membrane in the unmade pouch and make the pouch around the membrane by smooshing double-folded parafilm edges with the spatula tool. leave the top open, pipette in 1mL blocking buffer into the pouch, set aside and watch for leaks. Make the other pouches the same way. Dilute the antibodies need to look up how much i added, v imp! 1mL blocking buffer. Confirm that none of the pouches have leaked. Add the antibodies to the appropriate pouches, LABEL each pouch with the antibody. Seal the tops, rotate at 4degC overnight.
Antibodies

H3-ab1791 Rb pAb from abcam
New - Millipore rabbit poly 07-449 (purchased this month)
Old - Millipore rabbit poly 07-449 (purchased last year)