Use the Invitrogen TOPO TA Cloning kit. Please read the instruction manual carefully before starting. Invitrogen has changed the kit several times in the last year. The latest manuals are versions J and K as of 9/00. Please note that the kits no longer contain b-mercaptoethanol or stop solution but now contain a salt solution which is added to the cloning reaction.

The TOPO vector has an overhanging 3' T. Your PCR product must have an overhanging 3' A. Be sure to include a final extension step of 7 to 30 minutes during PCR. Longer PCR products will need longer extension times.

It is a good idea to purify your PCR product on a Qiagen PCR purification column before cloning to get rid of your PCR primers. Otherwise you may get a lot of clones that only have your primers as inserts.

The cloning kit has 2 parts: 1) A box containing the vector and salt solution which is stored at -20C and can be found in the enzyme freezer. 2) A box containing the competent cells, SOC, and pUC18 control DNA that is stored at -80C and is in the bottom compartment of the freezer next to the gel room. Handle the competent cells with care. Thaw them on ice and only thaw them when you are ready to use them. If they warm up they will not transform. The SOC is very rich medium. Be careful not to contaminate it or you will contaminate everyone else's transformation. Do not use it if it is cloudy or has anything growing in it. Return it to the freezer as soon as possible.

Before you start the transformation you need to do several things: 1) Warm a waterbath or heat block to 42 degrees for the heat shock step. 2) Thaw the SOC and let it come to room temperature. It's best to thaw it on your bench. It's more likely to get contaminated or knocked over if you put it in a waterbath. 3) Make sure you have X-gal and Amp plates. The X-gal is in the sequencing box on the top shelf of the -20 freezer at the end of the lab. It is 50mg/ml in N,N-dimethylformamide. Amp plates are made by the kitchen and are in the coldroom at the end of the lab. You may want 2 plates for each transformation. To ensure a good density of colonies you can plate 50ul on one plate and 200ul on the other. Dry the plates in the 37C incubator with the lids ajar for at least 15 minutes. Spread 40ul of X-gal on each plate and keep at 37C for at least 30 minutes. Leave them there until you are ready to plate.

The new manuals have some very vague instructions so here are some suggestions: 1) For the cloning reaction the instructions say to add 0.5-4ul of PCR product, but no concentration is given. Most people use a total of 20ng. Incubate at room temperature for 5-10 minutes. 2) For the transformation, the instructions say to incubate on ice for 5-30 minutes. Twenty minutes works well. 3) After adding the SOC, the instructions say to incubate at 37 degrees for one hour. People have found that 30 minutes is sufficient. Incubation is done in the roller drum in the 37 degree incubator.

The TOPO kit contains a very useful quick reference card to use while you are transforming. A card is taped to the front of the enzyme freezer. Please return it when you are through. You should do 2 controls:1) vector alone. It should give you very few colonies. The vector should not be able to self-ligate. 2) pUC18 to check transformation efficiency. See instructions on page 10 of the manual.

After plating, incubate the plates overnight at 37 degrees. Remove them the next morning and store at 4C until ready to pick colonies.

II. Preparing DNA

There are 2 ways to prepare your DNA for sequencing: minipreps and PCR. The PCR method is faster and easier but occasionally people have found that it does not work well on their construct. Whatever method you choose, if you've never done this before, I suggest you do a few samples and send them for sequencing to see if you've got the method working before doing 100 at $7-10 a sequence. Also we have a Qiagen vacuum manifold (on Pam's bench) that can be used for both methods if you have a large number of samples. It uses strips of 8 column filters and processes 48 samples at a time, so it is best to do your samples in multiples of 8.

PCR method

Decide how many sequences you want to do. You will need to pick 50 to 100% more colonies than you need because some will not work and some will be the wrong size.

1) Make up the appropriate amount of 5xPCR master mix. You will need 20ul for each clone.

Stock Resultant

PCR Buffer 300ul 10x 5x

JP21.145 F primer 60ul 100uM 10uM

JP21.144 R primer 60ul 100uM 10uM

dNTPs 120ul 5mM(ea.) 1mM(ea.)

Water 54ul

Taq 6ul Non-commercial

Total 600ul

2) Fill enough 0.2ml strip-tubes or PCR plates with 80ul of water to accommodate all your colonies. Pick a white colony with a 10ul pipet tip. Place the tip on the end of your P10 pipettor and expel the colony into 80ul of water by pipeting up and down 5 times. Heat in PCR machine on INSTANT INCUBATE at 95C for 5 minutes to lyse the cells.

3) Add 20ul of master mix to each tube. Pre-heat to 94C for 4 minutes, then do 20 cycles of (92C for 1 min, 50C for 1 min, 72C for 1 min).

4) To check the quantity and quality of your DNA, run 3ul on a 2% agarose gel along with 4ul of Gibco low DNA Mass ladder. It is not necessary to run all your samples. A few of each type will do.

5) If you just have a few samples, purify using the Qiagen Qiaquick PCR purification kit and follow the directions in the handbook.

If you have a large number of samples, use the Qiaquick-8 PCR purification kit and the vacuum manifold. It's best to have someone help you the first time you use the manifold. However, a few notes: 1) Apply,turn off, and release the vacuum slowly; otherwise your samples will spray everywhere and cause cross-contamination.

2) To elute your DNA, place the plexiglass block in the bottom of the manifold. Place an ISC 96-well plate on the block and the top of the manifold holding the columns over the plate. Make sure the columns and the wells are aligned and that the tip of the column is below the top of the well. Add 80ul of elution buffer to the center of the column. Let stand for 5 minutes and then turn on vacuum for 5 minutes. Cover with a microseal film for storage.

6) Run 3ul of all your samples on a 2% agarose gel and quantitate.

There are several facilities you can use. I will list them in order of preference.

1) The Biopolymer Facility at Harvard. Web address: BPF.med.harvard.edu Cost: $10/sample. Advantages: very good quality of sequence, courier service picks up and delivers samples (Call Doug at 257-5754), has standard primers that they provide at no extra cost. Disadvantages: Not high-throughput. They think 30 samples are a lot. However, not really much slower than the other places.

Amounts of DNA and primer required: They call primers they provide " standard"

and ones you provide "custom". For some reason they ask for different amounts of template to use with the 2 types of primers.