Colony Hybridization Protocol 3-15-12

2015-01-13

azim58 - Colony Hybridization Protocol 3-15-12

Colony Hybridization Protocol

Approximate timing of experiment

o Lift colonies -> Incubate 30 min -> Add IPTG -> Incubate 3-4 hr
-> Lyse bacteria (2-3 hours) -> Block (30 min or overnight) ->
Primary (1.5 hr) -> Secondary (1.5 hr) -> Scan with Typhoon (30 min)

Materials

o 10 mM IPTG
- 20 mL IPTG into 180 mL Carb
- powder last seen at -20 C on shelf of door
- 238.3 g/mol
- for 50 mL 10 mM IPTG
@ 10*mmol/(1*L)*1*mol/(1000*mmol)*238.3*g/(1*mol)*1000*mg/(1*
g)*0.05*L = 119.2 mg IPTG in 50 mL H20

o Lysis Buffer

@ 200 mL solution
# 197 mL 1X PBS
# 1% triton X-100 (so 2000 uL)
* last seen on Felicia's shelf

# 1 mM PMSF (a protease inhibitor) (so 1000 uL of 0.2 M PMSF
* last seen DNase box 3rd shelf in -20 C (powder last
seen in back chemical cabinet under "P")
* PMSF stands for phenylmethanesulfonylfluoride or
phenylmethylsulfonyl fluoride, and it is a serine protease
inhibitor

# 20 complete protease inhibitor tablet (add tablet just
before use)
* last seen at 4 C fridge by door on JC's shelf

o Lysozyme
- 10 mg lysozyme in 5 mL PBS for multichannel (or 60 mg for 30
mL)
@ last seen 4 C fridge

o DNAse and MgCl2

@ Example preparation: Add 35.6 uL of 0.2 mg/mL DNAse and 400
uL 1 M MgCl2 to 400 mL 1X PBS
@ DNAse (final concentration desired 0.0178 ug/mL)
# 200 ug/mL prepared with 1 mg in 5 mL RNAse free H20
* last seen -20 C in 15 mL tube

# 2.5 ug/mL prepared with 375 uL of 200 ug/mL in 30 mL H20
# powder last seen -20 C Q freezer in door in small bottle

@ MgCl2 (final concentration desired 1 mM)
# 1 M liquid solution last seen on Felicia's shelf
# comes as MgCl2*6H20 which is 203.3 g/mol (powder last
seen on chemical shelf in back)
* Example calculation:
10*mmol/(1*L)*1*L/(1000*mL)*15*mL*1*mol/(1000*mmol)*203.3*g/(1*mo
l) = 0.030 g
* last seen -20 in 15 mL tube by DNAse)

o LB/carb
o EDTA (final concentration desired (1 mM))
- 500mM*y/200000uL=1mM
- last seen on chemical shelf by weights

o LB Plates (for titrating to determine glycerol stock concentration
o Bioassay tray (245X245 mm^2)
o Blot paper for drying
o nitrocellulose blotting membrane for lifts

Plate serial dilutions of glycerol stock onto a large 245X245 mm^2

bioassay tray (perhaps 2 10^(-2), 2 10^(-4), and 2 10^(-6) dilutions).
o Spread about 382 uL (derived from:
50uL/(pi*50mm^2)=y/(245*245^2mm^2) -> 50uL/7845mm^2=y/60025mm^2 -> y
= 382uL)
o Approximate Concentrations of Tumor cDNA Library Glycerol Stocks
from past experiments can be found at that link
o Make sure to return glycerol stock to -20 C

Gently apply the nitrocellulose filter onto the plate with colonies

to "lift" the colonies. Don't press too hard on the filter to avoid
damaging/smearing/removing the original colonies. Puncture a pattern of
holes through nitrocellulose into LB plate with colonies so that these
holes can be used to align the final nitrocellulose image with the
actual colonies on the plate later on to identify specific colonies
(perhaps puncture a square pattern of holes on top and a triangle
pattern on the right). Place plate back into 37 C so that colonies can
be picked the next day.

Place the colonies into a container containing 180mL LB/carb
Incubate with slow shaking (25 rpm) at 37 C for 30 min.
Add IPTG to LB for a final concentration of 1 mM

o Example Calc: (10mM*y/200*mL=1mM -> y=20mL of 10mM IPTG in 180mL
LB/Carb

Incubate the filter for approximately 3-4 hr 37 C 25 rpm
Prepare lysis buffer (probably 200 mL). Note that it is probably best

to use one lysis buffer solution for a whole batch of plates so that
the minimum amount of protease inhibitor tablets and other reagents is
used.

Drain away LB in container. May want to rinse container and filter

several times with PBS. Add lysis buffer (probably 100 mL)

Add 2.5 mL of lysozyme solution
Incubate at room temperature 15 min with occasional shaking
Add DNAse/MgCl2 solution
Rotate gently at 4 C 1 hr. This can be done with a thermomixer,

rocker, or rotator in 4 C room. Discard solution

May want to add EDTA to prevent bacterial growth on membrane if it is

going to be stored (final concentration 1 mM)

Incubate RT or 4 C with gentle shaking 15 min. Discard solution.
Block filter with 1% BSA in TBST 30 min - 2 hr (or overnight at 4 C

if desired). Use a platform shaker to gently swirl the solution.
Discard BSA/TBST solution.

Add primary antibody diluted 1:1,000 in 5% BSA in TBST for 1-2 hr at

room temp. You will probably want to save this primary antibody
solution afterward.

Wash 3X with TBST (may want to wash on rocker for 5 min intervals)

(try not to add TBST so harshly that it washes some colonies away)

Apply direct labeled secondary antibody (often goat anti-mouse

AF647). Dilute biotinylated secondary antibody 1:10,000 in 5% BSA in
TBST and incubate 1 hr RT with shaking. You will probably want to save
this secondary solution afterward.

Wash 3X with TBST (may want to wash on rocker for 5 min intervals)

(try not to add TBST so harshly that it washes some colonies away)

Dry the filter between a sponge filter
Image with Typhoon

o Turn on the computer for the Typhoon and select the Typhoon
Scanner icon.
o Set to fluorescence
o Click Setup
o Deselect laser options
o Select the appropriate Emission and excitation lasers
o Last Settings used were: Excitation: 633 nm, Emission filter: 670
nm, Dye: AF647
o May want to reduce the PMT to 350
o Orient membrane on scanner
o Select blocks to scan and click "scan"
o Ensure to save file on server or flash drive instead of on the
computer or the file will be deleted later. Note that the standard
.gel files do not open well in other programs so make sure to at
least save a .tif file.

Once colonies are selected, pick them from the plate inoculate them

into 1 mL culture 37 C 250 rpm overnight (may be best to inoculate in
afternoon rather than early morning).

Perform plasmid miniprep
Sequence plasmids (Sequencing Protocol)

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Other documentation
Initial Protocol from Mara 3-12-12