Overview :

The mbuna and tilapia libraries have been selected to contain CA repeats and those DNA fragments are now cloned into the TA cloning vector pGEM-T. E coli needs to be transformed to contain the plasmid vector and plated on agar plates. After overnight culture of the plates, there will be individual colonies which can be picked into 96 well plates of LB and grown for a few hours. This culture can be used in a PCR test using two PCR primers contained in the vector (T7 and M13R) to determine the insert size. The ones which have insert sizes >200 bp will most likely contain inserts with CA repeats and are our positives.

We then go back to the 96 well plates and grow up larger overnight cultures of the positives. These can then be miniprepÕd to extract the plasmid DNA from the E coli cell culture. It is best to get all the positive colonies through the miniprepping as soon as possible as the resulting plasmid DNA is the most stable form for the clones.

The plasmid DNA is sequenced in one direction using the T7 primer. For many of the clones, this gives sufficiently good sequence that either the locus can be thrown out for some reason (repeat too short or no front sequence) or the locus is defined and primers can be designed. For 30% of the clones, they need to be sequenced in the reverse direction (the sequence sometimes dies in the repeat) so that we get good sequence on both sides of the repeat.

Once the clones have been sequenced, they can be entered into a Filemaker or MySQL database. Primers can then be designed for ones with sufficient flanking sequence on each side and microsatellites with greater than 12 repeats. The primers are then ready to be ordered and tested.

A few numbers based on the tilapia library results to date:

# of transformations	3
# of clones picked	700 (we picked a lot of blue ones Š see below)
# of clones w/ good inserts	375
# of loci after seq w/ T7	110 (i.e. just forward sequences)
# requiring reverse seq	80
best guess of # loci	175

The details

Things to prepare ahead:

LB amp plates (keep at 4 C)
LB broth with amp (keep at 4 C)
Ampicillin (4 mg / ml, filter sterilized and kept in freezer).

Step 0. Prepare LB plates (several days ahead)

Prepare LB agar plates with ampicillin. Make 1 l of LB agar. Autoclave (takes about 45 min). Watch autoclave so remove agar just after it is done (if agar cools below about 40C it will solidify). Put in 50 C water bath (check its water level ahead) to cool to this temperature. If agar is too hot when ampicillin is added it will be inactivated. After about 15 minutes, check. Add ampicillin to 50 ug/ml. (Typically add 12.5 ml of 4 mg/ml stock ampicillin). Swirl well to mix but try not to introduce lots of bubbles.

Pour into each of about 40 round plates so that agar is 3-4 mm high. DonÕt need too much. Let cool to set. Stack and store upside down at 4 C. Can label plates as LB amp using blue and red stripes.

Step 1 : Library transformation and plating (Days 1 and 2)

For the first plating it is probably best to plate 2 plates with the transformation mixture and save the rest the next day after the plating density has been checked.

a) Prep plates - In morning, put needed number of plates in 37 C oven. As with all plates, put in upside down so agar surface is inverted. This allows plates to dry. If they are noticeably wet, they can be tipped open to increase air flow (but still kept inverted).

Before starting the transformation process, add X-gal and IPTG to plates. Typically make master mixture of 40 ul X-gal (20 mg/ml) and 8 ul IPTG (100 mg/ml) for each plate. Add to plate and spread with sterile (flamed) spreader.

b) Transformation - The pGEM-T ligation mixture is either

chemically transformed into JM109 E coli cells following the Promega protocol or
electroporated into DH10B cells

Chemical transformation

Prior to starting check that there is a 42 C water bath (can use the autogene in a pinch). Essentially, add 1.5 ul of library ligation mixture to 50 ul of cells. Mix and place on ice for 20 min. Put in 42 C water bath for exactly 45 sec. Put back on ice for 2 min. Add 950 ul of SOC (which need to thaw ahead). Shake at 37 C in shaker (150 rpm) for 1.0 hour (The protocol says 1.5 hours but this only increases time to make duplicate clones, so keep it shorter).

Electroproation

Prior to starting, thaw a 1 ml aliquot of SOC. Chill a 1 mm gap cuvette on ice (can use 2 mm cuvettes also). Get a 40 ul aliquot of DH10B cells from -80 C freezer and put on ice to thaw. Add 1 ul of ligation to cells. Transfer to electroporation cuvette. Transform at 1.8 kV. Immediately after electroporation add 950 ml SOC. Transfer to a 15 ml snap cap tube and shake for 1 hour.

c) Plating - Take plates from oven. Add 50 ul of transformation mixture to first plate. Spread with sterile spreader. Add 100 ul to 2nd plate. Spread with sterile spreader. Return to oven, placing them inverted. Leave overnight. Plating is best done late in the afternoon so the plates do not incubate for more than 12-16 hours.

d) The rest of the transformation can be put in the refridgerator. Based on the cell densities the next morning, the rest of the transformation can be plated on X-gal/IPTG prepÕd plates. We often end up plating about 100 ul per plate. You want the colony densities such that there are only single colonies but their densities are high enough that you have about 50 colonies per plate. The colonies which contain inserts will be white or light blue. Most of the dark blue colonies do not contain an insert. We found that some of these did have inserts. However, it is only about 10-15% of the dark blue ones which may not be worth the extra effort of screening. Last time we waited >1 week to plate the 2nd part of the transformation and these plates had lower cell densities. If we plate it the next day, we should get sufficient numbers of colonies to skip the dark blue ones.

Step 2 - Colony picking (days 2 and 3)

Colonies will be picked into LB broth which contains ampicillin to same 50 ug/ml concentration. The broth is autoclaved and then amp is added. LB broth can be stored at 4 C for several months.

Fill a 96 well plate with 60 ul LB per well. Using P-20 with clean yellow tip, pick a single colony and swirl it in a well. Pipet up and down several times to mix. Once the plate is all innoculated, cover it with lid and put in 37C oven for several hours to grow. When done, seal the plate extremeley well with sealing tape. Check all the edges for air leaks as this will lead to the plates drying out and dead cells. Store the plates at 4C.

Step 3 - PCR test of insert size. (few days or weeks)

For each well culture, set up a 25 ul PCR reaction to test for insert size. The T7 and M13R primers flank the insert site. The resulting PCR product can be run on an agarose gel to size it.

a) PCR reaction Š Make a master mix. Add it to a 96 well plate. Then use multichannel pipettor to add the cell culture (can also do on Biomek). The strip caps work better than sealing tape (though are more expensive) so that individual rows can be handled separately.

PCR mix

10X buffer	2.5 ul
dNTP	0.5
T7	1
M13R	1
Taq	0.15
H2O	17.85
Cell culture	2
Total	25

PCR conditions : 94 20s / 50 45 s / 72 1 min 35 cycles.

We found you can set up all the PCR reactions on one day and put them in freezer. Then just keep loading them into the PCR machine. This did produce double bands on the gel, but the smaller band corresponded to the correct size. The larger bands were about 2x the smaller bands in size?

b) Agarose gel - The large gel rig can be used to run one 96 well plate at a time. Use 1% Seakem in TBE (I think about 250 ml). Add Ethidium bromide (5 ul / 100 ml). When pouring the gel, make sure to cool the agarose or the gel plate warps and the end plates leak. Use three 42 well combs placed in the top three slots (leave 4th slot empty).

Spot parafilm with a 2ul spot of orange G loading dye for each well. Take up 5 ul of PCR mix and mix on parafilm with loading dye. Load into well on gel. Using a new box of tips and keeping the tips, loading dye spots and PCR wells in registry helps keep it all straight. Leave spaces between the columns so can see sets of 8 on gel. This means load 4 sets of 8=32 wells in each of 3 rows of wells (eg. Plate columns 1-4 in top row, 5-8 in next row etc). Since there are 42 wells, there are plenty of left over spaces to spread between the column sets.

Load size standard in empty lanes. Probably 2 size standard lanes per row are sufficient. Using the OX/HaeIII size standard may be easiest as it has small sizes which can be cleared from the gel. However, 1kb ladder has nice 500 bp band for choosing clones > 500 bp.

It is easy to load one gel and let it run halfway to the next comb. Take itÕs picture. Then load another plate loading the size standard in new wells. After this, it is probably best to run the gel clean before running another set. Adding EthBr to end well helps keep from depleting the EthBr from the gel.

Run the gel for about 1 hour at 110 V. The orange G dye runs at the smallest size so it is a good guide for when you will reach the next row of wells.

The size of the PCR band includes some vector and linker sequence. For the T7 and M13R primers plus linkers on each end of the PCR insert, this is about 270 bp. Therefore, insert size = PCR band size Š 270 bp. If you enter the PCR band size into Filemaker data base on the Ņmore screening info pageÓ, the database will automatically subtract this number to give you insert size. The size which is listed on the main database page is this insert size.

Based on this data, determine which clones have insert sizes > 200 bp (or PCR band sizes >500 bp). These are the positives to be minipreped.

Step 4 - Miniprepping the plasmid DNA

Each of the positive clones need to be grown up in an overnight culture and then mini prepped (using Qiagen kit) to extract the plasmid DNA from the E coli cells. It is easiest to deal with one 96 well plate at a time. Each plate typically gives about 50 positives to be miniprepÕd (though there may be more if we pick fewer dark blue colonies). 100 minipreps is about all I can handle in one day. Alternatively, the positive clones could be transferred to a new 96 well plate, grown in deep well blocks overnight and prepped with the Millipore Montage kit by hand or on robot.

a) Overnight cultures - Late in the day, set up round bottom snap cap tube for each colony to be grown and label it. Add 3 ml of LB broth with ampicillin. This is easiest using a 25 ml pipette and the electronic pipettor kept in the sequencer lab. Add 10 ul of the culture from the 96 well plate to the side of the snap cap tube. Put on the cap and invert to mix the culture in. When all the tubes are ready, put them in the shaking incubator at 37C and 200 rpm. Leave for 12-16 hours though 12 hours seems about ideal. The tubes can then be removed and put in fridge. It is best to miniprep them that next day though I have miniprepÕd up to 2 weeks later.

b) Minipreps - Use Qiagen miniprep kit (easy directions). Pour 2 ml of O/N culture into a 2ml tube. Spin for 5 minutes to pellet cells. Pour off LB. Pipette off last bit of LB. Add cell resuspension, then lysis buffer and finally neutralization solution. Spin. Transfer supernate to filter. Spin. Wash with buffer. Spin and spin again after disguarding flow through. Elute with 50 ul of water i nto clean labeled tube. Keep in freezer. You donÕt want the DNA to sit too long in the various buffers. It is best to take one set of 24 all the through and then start another set. However, I was able to run two sets of 24 alternating between them at the different steps and that worked OK.

c) Quantify the plasmid DNA concentration using the fluorometer. Enter the concentration in Excel spreadsheet. Want 500 ng for a sequencing reaction. Determine volumes of plasmid and water for sequencing so plasmid + water = 11 ul. IÕve had some luck with lower concentration plasmids though not consistent luck. Nothing less than 10 ng/ul works so forget those.

Step 5 - Sequencing

a) Cycle sequencing. Use Amersham sequencing kit to sequence each clone using 500 ng of plasmid DNA (or as close as can get to 500 ng) and T7 primer. If concentrations are above 100 ng/ul, you can use half reactions and so save reagents.

Reactions are

b) Sequencing gel. Resuspend pellets in 2.5 ul of Amersham loading dye. Load 1.2 ul per lane. Run 48 lanes per gel. Filter set A works nicely though filter set E can be used if need to run on gel with ABI dyes.