Typical Cloning Strategies (Beth 1/02)

From one vector to another vector:

1) Although some people might argue I always start by making a Maxiprep or Tip100 of each vector I will be using- the parent vector and the recipient vector. That way if at first the ligation doesn’t suceed, I have enough DNA in the freezer to try try again. Otherwise you waste a few days doing another DNA prep.

2) Some Quiagen Maxiprep tips: Once the supernatant from Step 14 (70% wash) is decanted, mark the place where the pellet is and keep the tube inverted for 5-10 minutes. Carefully wipe the sides of the tube with a Kimwipe staying far away from the pellet. Add 300uL TE to the side where the pellet is and using a slightly cut 1000mL pippette tip resuspend the pellet and wash the sides of the tube gently. Transfer this to an eppendorf tube. Add 1/10 volume of 3M NaOAC pH 4.6 and mix. Add 3 volumes of ice cold 100% EtOH to the tube (900uL) and mix- you should see bubbles or white snot form in the tube when you invert the tube to mix. (If you don’t see this, place the tube in the –80°C freezer for 15 minutes prior to the next sentence). Spin in a table top eppendorf fuge at 4°C for 25-30 minutes. Decant supernatant, mark where pellet is and rinse the tube with ice cold 80% EtOH (some people use 70% but 80% is more careful about keeping the pellet since there is less water content.) Do not resuspend the pellet. Just rinse the tube sides by flipping the tube once or twice and then invert to dry for about 15-25 minutes. I refuse to use a speedvac but you can at your own risk of sucking the DNA into la la land. The pellet will turn from a white opaque color to clearer. Resuspend in 100uL TE and measure the OD260 and OD 280 at a dilution factor (D.F.) of 1:100. The OD 260x50ug/mLxD.F. will give you the amount of ug DNA per mL- change this to microliters for ease later. FYI RNA is 40ug/mL and Oligos or single stranded DNA is 33ug/ml factor X OD 260. Your 260/280 ratio should be 1.8. Too low and there is probably protein in it, too high and there is probably genomic or RNA in it.

3) Once you have maxipreps of each piece of DNA set up some digestions. Make sure that you check the temperature and BSA requirement, buffer compatibility for double digests and efficiency of cleavage. I usually put all this pertinant information from the New England Biolabs catalogue in my 3 ring binder for ease of access.

For a typical cloning procedure, I usually cut 1-2ug of vector containing the desired insert and 1-2ug of vector that will receive the insert. This is a step where you will get all sorts of suggestions and you will just have to see which way consistently works best for you. ((I used to do it differently (with a very small amount of vector compared to insert amount. You can actually determine different ratios to use with the OD). Typically I used to dilute the recipient vector 1:50 or mote and use 1uL of that with 1uL, 3uL, or 5uL of insert – but I will describe this in the ligations section)). One thing to note is that if a ligation reaction has greater than 1ug total DNA it will not work too well. The ligase is inhibited with too much DNA around. The reason I start with 1-2ug each is that in the DNA purification steps you tend to lose DNA.

4) For most digestions I use a 25uL reaction. This can fit all in one thick lane on a DNA gel when you purify it later. Therefore use 2.5uL of the 10X buffer, 1uL of each enzyme, add the equivalent of 1-2ug DNA and q.s. the volume to 25uL with H2O. If I am using an enzyme with star activity (like Kpn1) then I use 1uL of enzyme in a 100uL total reaction and just load a bunch of lanes on the DNA gel later. Digest for a MINIMUM of 3 hours in a 37°C water bath or on the bench top for 25°C enzymes. When you are screening for positive clones you can digest shorter but for cloning you need to be SURE it is completely digested prior to using it in a ligation. If you do an overnight digestion use half the amount of enzyme since there is some paranoia about nucleases and abductions….although I have never had a problem. ALSO check the halftime of the enzyme at 37°C. There is no use adding all BamHI, for example, at once since the half-life at this temperature is 1 hour. Instead start with less and Spike as you go. To note: many R.E.s also have star activity when there is too much glycerol present, always calculate the total volume of your digest so that there is never more than 10% glycerol total at any time.

5) After the Digestion, heat inactivate the enzyme if this is possible. Add DNA loading dye (Maniatis) to 1X and run on a gel (see Maniatis for the percent gel to run in order to see your fragments the best (Usually 0.8-1% is fine). I put a small amount of Ethidium in the gel itself after microwaving it to melt it into solution but you have to have special ethidium waste for it. Run a DNA ladder along with this- the 1kb marker from NEB is particularly lovely.

6) Cut the bands out, but cover the sections from the UV light until you are ready to cut the band out with a razor blade. Bring some eppendorfs along with you to the UV box and place the cut out bands into their tubes. Then use Quiagen Quiaquick kit to purify your gel bands away from proteins and salts prior to the ligation. There are two methods you can use 1) Modifications to Quiaquick that improve your ligation: In Step 7 bind all of your insert to a column by centrifuging it through. Then centrifuge all of your vector through the same column so that both your insert and vector will “see” each other through the process and it will eluted in a smaller volume for the ligation (17uLEB instead of 50uL). After adding 17uL to the CENTER of the column, place in a dry 50°C heat block for 1-5 minutes and spin to collect DNA. Store DNA on ice and get reagents for ligation ready. For ligation add 2uL of NEB 10X T4 ligation buffer and 1uL of T4 ligase and pippette to mix. O/N at 16°C (we have a heat block in the cold room set so it maintains a temp of 16°C. or you can use a PCR machine. Or in Erika’s lab I used to put my tube in an ice bucket of 16°C water and seal it tight with a lid and float my tube in it in the cold room. The temperature will come down by 8 hours but the ligation is often done by then anyways.

2) The other method, RAPID LIGATION, I like much better:Quiaex purify and elute in 20uL of EB. Often it doesn’t. Ifound that it will always come out (provided your insert is within the proper size insert for the kit) if you elute with 20uL heated EB at 50°C, or if you heat the tube with the EB in it for 5 minutes at 50°C. Then spin as normal. The next step you can be lazy about its not worth skipping, because if you do not check to see if you have DNA and proceed forward with the ligation and transformation and you had no idea of how much insert (if any) or vector you had, then you are a poor experimentalist. Mol Biol is like any other experiment. You need to be sure of the quality and quantity of the reagents, otherwise, do not even bother doing the experiment. Run out 2uL on a gel to be sure something came out. Use the B-M Rapid ligation kit. From this point I take, insert, vector and water and add together for a total volume of 8uL, I add 2uL of 5X DNA dilution buffer #2 from the Rapid Ligation Kit. Add 10uL of 2X ligation buffer #1 (if the kit is out, then use the normal 10X T4ligase buffer stock and make yourself some 2X ligation buffer.) You can also use T4 ligase if the rest from the kit is gone. The only thing special about this kit is the DNA dilution buffer which has a crowding agent like PEG in it. Add 1uL T4 ligase, leave on bench for 5-10minutes. Then back on ice. Transform or freeze until you have time to transform.

7) Transform 10uL of a ligation into DH5alpha or equivalent competant cells. To transform see my other protocol. DH5 alpha cells do not make as large of colonies as BL21 and do not grow as fast, usually if I plate by 6pm then