Insert preparation
1
Insert PCR
NB: for each insert, amplify at least 4x 50ul PCRs for each strain so you have
enough at final stage for ligation.
PCR mix:
5ul buffer
5ul dNTP
3ul MgCl2
2.5ul primer F
2.5ul primer R
0.25ul TaqGold
11.25ul dH2O
30 ul total
plus 20ul DNA (1/500 dilution of either AJ or C57Bl6 DNA)
PCR amplification:
use rubber mat and heated lid on PCR machine to prevent evaporation and amplify with
Gold55 program (95oC 12 mins; 95oC 30 secs, 55oC 30 secs, 72oC 90 secs (for 35 cyles);
72oC 5 mins)
Pool together the PCRs of the same strain and amplimer so you have a total of ~200ul
PCR. Check 3ul of pooled PCR on check gel to: 1) make sure the product size is correct
for each amplimer, 2) to make sure that you have enough product to continue through
purification process, and 3) to ensure you don’t have non specific amplification product.
If there is non-specific amplification you will need to gel purify the specific band from
the other bands using a 1% low melt agarose gel. (see vetor preparation – gel extraction
for protocol. NB, this needs to be done only once for insert bands not twice as described
for the vector prep)
2
PCR purification
This is done using the QIAGEN PCR purification kit as per manufacturers instructions:
 Add 5 volumes (in this case 1ml) of buffer PB to pooled PCR from step 1 (in this
case 200ul)
 add in two 600ul lots to a spin column (1 per strain per amplimer)
 centrifuge at 13000 rpm for 60 seconds, and discard flow-through
 add 750ul buffer PE (make sure ethanol is added before using) to column
 centrifuge at 13000 rpm for 60 seconds, and discard flow-through
 centrifuge at 13000 rpm for an additional 60 seconds to dry column
 place column in a fresh labelled 1.5ml centrifuge tube
 add 50ul buffer EB to column, and let stand for 1 minute before centrifuging at
13000 rpm for 60 seconds to collect purified insert DNA
3
Klenow inserts
To each 50ul purified PCR product from step 2, add 2ul klenow enzyme, and 50ul
2xNEB2+66uMdNTP, and incubate for 30 minutes in a waterbath at 37oC.
For C) ligation control, add 1ug of stock ØX174 HaeIII digest and make volume to 50ul
and treat identically to other insert DNA.
(to make 2xNEB2+66uMdNTP, firstly make 10mM dNTPs by combining 5ul of each
dA-, dC-, dG-, dTTP stock (100mM) and 30ul dH20. Then make 2xNEB2+66uMdNTP
by taking 200ul of NEBuffer 2, adding 6.6ul of 10mM dNTPs, and 793.4ul dH2O)
4
PCR purification
This is done using the QIAGEN PCR purification kit as per manufacturers instructions
using a separate column for each amplimer and mouse strain:
 Add 5 volumes (in this case 500ul) of buffer PB to Klenowed PCR product from
step 3 (in this case 100ul)
 add in two 600ul lots to a spin column (1 per strain per amplimer)
 centrifuge at 13000 rpm for 60 seconds, and discard flow-through
 add 750ul buffer PE (make sure ethanol is added before using) to column
 centrifuge at 13000 rpm for 60 seconds, and discard flow-through
 centrifuge at 13000 rpm for an additional 60 seconds to dry column
 place column in a fresh labelled 1.5ml centrifuge tube
 add 50ul buffer EB to column, and let stand for 1 minute before centrifuging at
13000 rpm for 60 seconds to collect purified insert DNA
5
Kinase inserts
Take elution products from step 4, and to each tube add:
6ul 10x PNK buffer
1ul PNK enzyme
3ul dH2O
60ul total
incubate for 30 minutes in a waterbath at 37oC.
heat inactivate by incubating for 10 minutes in a heat block at 65oC
6
Ethanol precipitaton
To each tube from step 5, add:
150ul 100% Ethanol
6ul NaOAc
freeze on dry ice until solid, then centrifuge 20 mins at 13000 rpm. Remove supernatant
by pipetting, taking care not to dislodge pellet. Add 150ul 70% ethanol, flick mix, and
centrifuge 10 mins at 13000 rpm. Remove all supernatant by pipetting, taking care not to
dislodge pellet. Air dry pellet until completely dry (can speed this up by putting in 37oC
heating block for 10-20 mins). Resuspend pellet in 15ul dH2O
7
Quantitation
Take 1ul of product of step 6 and check on a 2% agarose gel, being careful to load 5ul of
molecular weight markers (1kb and ØX174 HaeIII) to use as a ladder for quantifying
amount of DNA in insert preparation. Also load 1ul of vector prep which will be used in
ligation, so you can get a good estimate of the quantities of each you need to ligate.
Vector preparation
1
Digest
10ul maxiprep vector DNA (~20ug)
4ul SmaI enzyme
2ul SAP enzyme (shrimp alkaline phosphatase)
4ul NEBuffer 4
20ul dH2O
40ul total
incubate at 25oC for 2-3 hours or overnight
load 2ul on check gel 1-2% agarose alongside the equivalent amount of uncut vector to
make sure digestion has worked and you can identify the cut band from the uncut band by
comparison with 1kb ladder
2
Gel extraction
Prepare 1% low melt agarose gel in small gel tray (total 200ml volume) by:
Weigh 2g low melting point agarose, and add to 200ml 1xTAE buffer and melt in
microwave on full power for ~4 mins. Pour into gel tray (don’t add EtBr yet) with one
large well comb (should fit 50ul sample in).
Add 10ul orange G to 40ul digest, and carefully load the whole amount on gel after
placed in 1x TAE running buffer, loading every second well to make sure you don’t have
sample spill-over. Also add 20ul 1kb ladder to identify “cut” band. pGL3-basic vector is
4818bp, but promoter vectors will be larger depending on size of insert (RGS18SNP5196
= 930bp)
Run gel in cold room during the day (at least 5 hours) at a maximum of 50-60V (gel will
melt and you will loose your DNA if you run it faster). Carefully take gel out from
running buffer and place in plastic lunchbox. Stain gel with EtBr (add 20ul to 200ul 1x
TAE or H2O) and pour over gel. Leave for 20-30 mins for EtBr to infiltrate gel. Take
photo of gel to see bands and how far gel has run.
When you can see separation from uncut band (if visible) or the bands have run for a few
centimetres through gel, excise bands from gel using low UV setting on transilluminator
in dark room (in room behind equipment galley). Using a clean scalpel, cut bands out of
gel and put them in fresh labelled tubes. Cut excess gel (containing no DNA) around
from the edges so the gel piece just contains the band of interest.
Pour a second 1% low melt agarose gel, and load the gel pieces from the first gel into the
wells of the second gel, and run in cold room with fresh 1xTAE running buffer overnight
at 40V to further separate the cut bands from the uncut bands.
Next morning, excise bands from gel as previously, and extract DNA from gel slices
using QIAGEN gel extraction kit as per manufacturers protocol.
Weigh gel slice in epp (calculate gel slice weight by subtracting weight of empty epp)
Add 3 volumes (100ug = 100ul) of buffer QG to epp with gel slice, and melt in heating
block at 50oC for 10 mins with occasional mixing (every 2 mins or so).
 Add a maximum of 730ul to spin column, adding volumes sequentially to the same
column if volume of a sample is greater than 730ul.
 centrifuge at 13000 rpm for 60 seconds, and discard flow-through
 wash column by adding 500ul buffer QG and centrifuge at 13000 rpm for 60
seconds, and discard flow-through
 add 750ul buffer PE (make sure ethanol is added before using) to column
 centrifuge at 13000 rpm for 60 seconds, and discard flow-through
 centrifuge at 13000 rpm for an additional 60 seconds to dry column
 place column in a fresh labelled 1.5ml centrifuge tube
 add 30ul buffer EB to column, and let stand for 1 minute before centrifuging at
13000 rpm for 60 seconds to collect purified vector DNA
3
Quantitation
Take 1ul of product of step 2 and check on a 2% agarose gel, being careful to load 5ul of
molecular weight markers (1kb and ØX174 HaeIII) to use as a ladder for quantifying
amount of DNA in insert preparation. Load alongside insert preps before using in
ligation step.
Ligation
We aim for a 10 fold excess of insert to vector for each ligation, so the amounts of vector
and insert are going to be dependant on your quantitation gels from your vector and insert
preps. You may need to dilute the vector to get a good concentration for ligations. The
following can be used as a guideline…
Ligation mix:
1ul vector (depending on concentration)
1ul T4 DNA ligase buffer
1ul T4 DNA ligase enzyme
4ul insert DNA (depending on concentration)
3ul dH2O
10 ul total
we also set up controls for the ligation, including:
A) – contains vector, ligase buffer, dH2O – but no ligase
enzyme or insert
B) – contains vector, ligase buffer, ligase enzyme, dH2O –
but no or insert
C) – contains vector, ligase buffer, ligase enzyme, dH2O and
control insert of ØX174 HaeIII (prepared with other
inserts with klenow, kinase and purification steps)
incubate at 16oC overnight in waterbath in cold room.
Transformation
Needs to be done in cold room, and all tubes and tips precooled to make sure cells are ice
cold until they are heat shocked. Preheat waterbath in cold room to 42oC. Preheat rescue
broth (2xYT or LB) to 37oC in waterbath.
0.25ul β-mercaptoethanol (from electrocompetent cell kit)
2ul ligation
50ul XL10 cells
add β-mercaptoethanol and ligation to side of precooled tube on separate spots so they
don’t mix until cells are added.
Add XL10 cells and plunge into 42oC waterbath for exactly 60 seconds, then plunge into
ice for exactly 2 mins. Then immediately add 400ul of warm rescue broth (2xYT or LB)
to each tube and put immediately in 37oC shaking incubator for about 1 hour.
Plating out transformed cells
Loosen cap and melt premade LB or 2xYT agar in microwave (or time making this up so
it arrives from autoclave on morning of transformation) until fully melted (10 mins at
70% power, mix, then 5 mins 50% power, mix, 4 mins 50% power… NB this tends to
explode in microwave without attention, which means lots of cleaning, and much less
usable media, so be careful to try and prevent this with regular mixing)
Place agar/media bottle in waterbath at 50oC for a few hours to equilibrate to this temp
before adding ampicillin antibiotic. (NB if the media is too hot to touch comfortably with
your hand, it will kill the antibiotic and you will not have selection of transformed
colonies.)
Take bottle of agar/media, ampicillin, 2 packs of 20mm x 100mm Petri dishes, p1000
plus tip to biohazard hood upstairs.
Add 500ul of 100mg/ml ampicillin per litre of agar/media, mix by inversion trying not to
make bubbles (as these will get in your plates), and lay out your plates without lids in
hood.
Pour enough to cover the base of each Petri dish and leave with lids ajar for 20-30mins to
dry a little.
Label up plates for each transformation, and spread 100ul (or 200ul and leave to dry for 5
mins) transformed cells onto plate with plate spreader. Place with agar side up in 37oC
incubator (behind hood) overnight.
Picking colonies
Take Petri dishes out of incubator and count colonies on each plate. Plates A and B
should have few colonies, and plate C should have many colonies if ligation has worked
and the vector background (purification of cut vector from uncut vector) is clean.
Count colonies on each plate, and pick them into dH2O followed by glycerol for PCR
screening.
Prepare two costar plates for every 100 colonies, one with 100ul dH2O in each well, and
one with 100ul 30% glycerol (in LB or 2xYT media) in each well.
Pick colony from plate using wooden toothpick and put in one well of H2O plate, and
then into the well at the same position in the glycerol plate. Record on plate layout sheet
which colonies with each insert you have placed in which positions on the plate.
You can use both ends of the toothpick to pick separate colonies, just be careful not to
touch the ends to prevent cross contamination of colonies.
Once you have picked all the colonies, transfer 5ul of colony H2O into another costar
plate and amplify using vector primers RV3 and GL2 to determine which clones have
inserts.
Insert PCR mix:
1ul Buffer
1ul dNTPs
0.6ul MgCl2
0.5ul RV3 primer
0.5ul GL2 primer
0.05ul TaqGold
1.35ul dH2O
5ul total
plus 5ul colony dH2O
Insert PCR amplification:
Add 15ul mineral oil to each well to prevent evaporation and amplify with Gold55
program (95oC 12 mins; 95oC 30 secs, 55oC 30 secs, 72oC X secs (for 35 cyles); 72oC 5
mins)
X = 2 mins 20 secs for RGS18SNP5196 promoter vector with enhancers
X = 3mins 30 secs for RGS2 or Brinp3 promoters
Run products on 2% check gel and identify which wells contain colonies with inserts of
the correct size.
Sequencing colonies
Identify which colonies have inserts of the correct size (different for each
enhancer/promoter combination) and do 50ul PCR reaction for sequencing template.
PCR mix:
5ul buffer
5ul dNTP
3ul MgCl2
2.5ul primer F
2.5ul primer R
0.25ul TaqGold
11.25ul dH2O
30 ul total
plus 20ul colony dH2O
PCR amplification:
use rubber mat and heated lid on PCR machine to prevent evaporation and amplify with
Gold55 program (95oC 12 mins; 95oC 30 secs, 55oC 30 secs, 72oC X secs (as before) (for
35 cyles); 72oC 5 mins)
purify PCR with MANU030 PCR purification plate on vacuum manifold and prepare
PCR product for sequencing (see sequencing protocol)
Prepare sequencing reactions with RV3 and GL2 primers, as well as primers used for
amplifying inserts (for orientation of insert) and internal primers from promoters (for
RGS18SNP5196 enhancer clones, you can use RGS18_int254R as the internal primer
which should give you complete sequence and orientation of the enhancer element).
The primers are in my freezer.. gene specific ones for promoters are in boxes labelled
with the gene names, and enhancer ones are in the enhancer boxes (as stocks or dilutions)