Protocol for Microsatellite Library
Screening
An adaptation by Marc and Jen based on an
adaptation by Tasha of a document from Kevin
!!!!!!!!!!!When
libraries are received, immediately store at –80!!!!!!!!!!!!
Since the library comes as 1 ml of a
20% Glycerol stock of transformed E. coli (DH5α) and we only wish to
plate out a portion of the library there will be a couple of extra steps in
this process.
It is better to leave the plates over
the weekend before using. We
suggest that you make day 0 a Friday, and day 1 a Monday.
1. LB broth: 10 mL of broth will be needed for plating bacteria
3mL
of broth will be needed per prep (~150 preps per library)
5 g
Bacto-yeast extract
10 g NaCl
**Stir until
solutes dissolve. Adjust pH to 7.0 with 5N NaOH (~0.2 mL). Adjust volume to 1 L
with nanopure water. Autoclave.**
2. LB-Agar:
~30mL of LB agar will be needed per 90mm plate.
Prepare at least 5 plates per library, plus a few extra.
Recipe for
LB-Agar (Sambrook et al. A-4):
Just prior to autoclaving, add 15 g/L bacto-agar to LB broth mixture.
Autoclave.
When cooled somewhat (approximately 50C), add 100mg/mL carbenicillin to make a final concentration of 150 ug/mL (i.e. add 1.5 mL Carb for every 1 L of LB)
Mix and pour into plates
Store at 4C upside down in somewhat sterile conditions.
For plate pouring tips see Sambrook et
al., A-4
3.
Reagents: Bacto-agar,
bacto-tryptone, bacto-yeast-found in dry reagent storage cabinets AFTER end
of alphabet.
IPTG, X-gal,
carbenicillin are in the –20oC freezer
in the box labelled “chinook microsatellite project”
Sterile glass
beads are aliquotted in 50 ml conical tubes, located in “Library Screening”
drawer.
Solid X-gal is in –20 freezer, enzyme shelf
Preparation of Reagents
(day 1):
1.
Two
plates will be needed per library. LB-agar
+ 150 ug/ml carbenicillin plates should be incubated upside down at RT/ 20’
(or however long is necessary to eliminate most of the visible condensation and
moisture from agar surface in a laminar flow hood with their covers ajar).
This helps to dry out the media so that it will absorb what will be put
on it next.
2.
Proceed
one plate at a time.
3.
Add
the following to each plate in order-
100 uL LB media
12
uL 20% IPTG
40
uL 20 mg/ml X-gal (DMF)
4
to 7 sterile glass beads.
Roll beads
around in covered petri dish until reagents are thoroughly spread over agar
surface. Discard beads after use.
Do not use the plates for at least ½ hr. due to the DMF. This time also allows
reagents to absorb into agar. These
will be the “Library Plates”.
4.
Chill
labelled 1.5 ml epi tube on ice (1 per library). Rest sterile spatulas on two 90mm petri dishes (do not have
to be freshly opened sterile packs) at –30 oC somewhere on a rack
where you have elbow room.
Procedure
(day 1):
5.
Retreive
the library tubes from –80C freezer. Place in ice block tube racks and proceed
quickly to the walk-in freezer.
6.
[Done
in –30 oC cold room] Mix the library bacteria gently
with a pipette tip or spatula. Steriley remove ~150 uL frozen cells (a little
more than ½ volume of a pea) from 1mL glycerol stock to pre-chilled 1.5 ml epi
tube. [Return to Lab]. Replace
Glycerol stocks immediately. Allow
150 uL aliquot of bacteria to thaw on ice.
Mix gently with sterile pipette tip by stirring.
7.
Add
100 uL to one plate, labeled accordingly, then add 4 to 7 sterile glass beads.
Gently roll beads around in covered petri dish until cells are thoroughly
spread over agar surface. Discard
beads after use. Repeat, using 50
uL onto another plate. Allow bacteria to absorb onto agar before inverting. Incubate @ 37 oC, overnight (O/N). In the morning,
store plates a 4oC
Preparation of Reagents
(day 2):
Two
grid-plates per library should be used to grow up colonies (~150 colonies).
LB-agar + 150 ug/ml carbenicillin plates should be incubated at RT/ 20’
in a laminar flow hood with their covers ajar.
This helps to dry out the media so that it will absorb what will be put
on it next.
Add the following to each plate in
order-
100
mL LB media
12 uL 20% IPTG
40
uL 20 mg/ml X-gal (DMF)
4 to 7 sterile glass beads.
Roll beads around in covered petri dish
until reagents are thoroughly spread over agar surface. Discard beads after use.
Do not use the plates for at least ½ hr. due to the DMF. This time also allows
reagents to absorb into agar. Afterward, attach a grid transparency to bottom of
each plate (can be found in “Library Screening” drawer).
Making additional marks on the plate with a sharpie to identify the
correct orientation of the grid may save you great headaches later.
These will be the “Master Plates”.
Procedure
(day 2):
8.
Pick a white colony from Library Plate with sterile 10uL and inoculate
one segment of the grid on a Master Plate (i.e. lightly touch the edge of the
colony and spread {not poke} onto agar of grid).
Repeat till you’ve picked the number of colonies you want.
Incubate O/N @ 37 oC. Store
at 4oC. [Kevin’s note: Bluo-gal does not give a good indication of
non-recombinant clones, even after several days at 4 oC.
Picking clones from the Master plate seems to be more productive in the
long run with X-gal than Bluo-gal.]
Preparation
of Reagents (day 3):
9.
Prepare PCR “Pre-mix” (for 1rxn)-
5.85
uL PCR-grade H2O
1.0
uL
10X PCR Buffer
0.45 uL 50 mM Mg2Cl
1.2 uL dNTP Mix (2.5 mM each dNTP)
0.75 uL 20 uM forward primer (pUC19seqF)
0.75 uL l 20 uM reverse primer (pUC19seqR)
10.00 uL total.
[pUC19
primers in –20oC freezer in box labelled “chinook PCR
reagents”]
10.
Prepare Taq
Mix (for 1 rxn)- (not added until step 16)
4.35
uL Pcr-grade dH2O
0.50
uL 10X PCR Buffer
0.15
uL Taq DNA Polymerase (do
not add till ready)
5.00
uL total
= 15 ul total reaction volume
Procedure
(day 3):
11.
Add
10 uL of PCR “Pre-mix” to each PCR tube. Inoculate each tube with a
“stab” from each clone from the master plate.
“Stab”= touch pipette tip to edge of colony.
12.
Incubate
clones in Pre-mix at 100 oC/10 min. Programmed in thermocyclers as
“kill”(this
lyses the
bacteria to release plasmid). Centrifuge
to collect; chill tubes on ice.
13.
Set up & pre-heat thermocycler(s) as follows:
1) 94 oC/ 4:30’ (X1)
2) 94 oC/ 0:30’
57 oC/
0:30’
72 oC/
0:30’ (X25)
3) 72 oC/ 2:00’ (X1)
4) 4 oC/
soak (X1)
This has been
programmed in thermocyclers as “msatscrn”.
14.
Add Taq to Taq mix listed in step 10 (above). Add 5
uL mix to each tube while on ice.
15.
Load
thermocycler- go read about organism of choice.
16.
68 samples can be run on an agarose gel
Make 2 L 1x TAE, pH 7.8 per agarose
gel-
80 ml 25X TAE Buffer
1920 ml dH2O
17.
Make 3% agarose gel (300 ml)-
9.0 g Agarose
300 mL 1X TAE Buffer
3 uL GelStar stain
18.
When cycler is finished, centrifuge products.
Dilute 6x loading dye 1:2 with water.
Add 10 uL of diluted Loading Dye to each PCR tube.
Load 1 uL PCR Rxn/loading dye per well.
Load 100 bp ladder in a 1:1 mixture with dilute Loading Dye.
19.
Select
those clones that produce a 250-800bp PCR fragment for 3 ml culture
andsubsequent purification and sequencing.
20.
Ensure
that there are plenty of sterile glass culture tubes, and sterile (non-barrier)
pipette
tips/toothpicks, 1 L & 500 ml containers containing 10% bleach. Nonsterile borosilicate tubes are in packages on storage shelves in thermocycler room. Caps are in drawers below balances. Autoclave tubes before use. Autoclaved tubes are kept in racks on high shelves above –20 freezer.
Preparation
of Reagents (day 4):
21.
Ensure
that incubator/H2O –bath is at 37 oC.
Incubator can hold 180 tubes.
Prepare
3 ml culture/clone: (LB + 150 ug/ml Carbenicillin)-
3 ml LB
4.5 uL 100 mg/ml Carbenicillin
Procedure
(day 4):
22.
In
the afternoon, use sterile pipette tip to pick white (recombinant) colonies from
the Master
Plate
for inoculation of 3 ml culture.
23.
Incubate at 37 oC, O/N, 300 RPM in incubator/H2O
–bath.
Procedure
(day 5)
23.
Centrifuge
tubes uncapped at 2000 RPM/10’. Make sure not to load tubes side by side, and
that
the tubes don’t touch anything when they rotate to a horizontal position.
Decant supernatant into 10% bleach.
24.
Process
3 ml pellets through Qiagen Plasmid Miniprep Kit (#27106). Pellets may be frozen
at
–20
oC if needed at this point.
26. Quantitate a dilution of plasmid miniprep via DNA quantitation program of FLORA fluorimager.