Ligation

Notes on preparation of DNA fragments for ligation.

1. Restriction digests:

Always cut a lot of your starting plasmids in a small volume; this will help in the gel purification of your restriction fragments by giving you a high concentration of DNA compared to agarose in your gel slice.. About 1-2 µg in a 20 µl reaction is good.

Restriction enzyme digestions are performed by incubating double-stranded DNA molecules with an appropriate amount of restriction enzyme, in its respective buffer as recommended by the supplier, and at the optimal temperature for that specific enzyme. The optimal sodium chloride concentration in the reaction varies for different enzymes, and a set of three standard buffers containing three concentrations of sodium chloride are prepared and used when necessary. Typical digestions included a unit of enzyme per microgram of starting DNA, and one enzyme unit usually (depending on the supplier) is defined as the amount of enzyme needed to completely digest one microgram of double-stranded DNA in one hour at the appropriate temperature. These reactions usually are incubated for 1-3 hours, to insure complete digestion, at the optimal temperature for enzyme activity, typically 37degC.

Protocol

1. Prepare the reaction for restriction digestion by adding the following reagents in the order listed to a microcentrifuge tube:

	sterile ddH20        q.s (where "q.s." means quantity sufficient)
	10X assay buffer     one-tenth volume
	DNA                  x ul (0.5-2 ug)
	restriction enzyme*  y ul (1-10 units per ug DNA)
		Total volume z ul
*If desired, more than one enzyme can be included in the digest if both enzymes are active in the same buffer and the same incubation temperature.

Note: The volume of the reaction depends on the amount and size of the DNA being digested.

Refer to the vendor's catalog for the chart of enzyme activity in a range of salt concentrations to choose the appropriate assay buffer (10X High, 10X Medium, or 10X Low Salt Buffers, or 10X SmaI Buffer for SmaI digestions). Restriction enzymes are purchased from Bethesda Research Laboratories, New England Biolabs, or United States Biochemicals.

2. Gently mix by pipetting and incubate the reaction at the appropriate temperature (typically 37 C) for 1-3 hours.

3. Inactivate the enzyme(s) by heating at 70-100 C for 10 minutes and store at -20 C.

4. Gel purifying DNA fragments: Purify all DNA fragments using Qiagen Gel Purification kit.

-When the gel is done, place it on a clean piece of saran wrap on a long wave (365 nm) UV box and take a picture. Make sure minimize the exposure to UV to avoid damaging the DNA. Cut the desired bands out of the gel with a clean scalpel.  Place the gel slice in an eppendorf tube. Can freeze the gel slice to store or proceed to Qiagen kit.

Manipulating DNA ends.

Blunting 5' overhangs: Add 1 µl 2 mM all four dNTPs to your 20 µl restriction digest. Add 0.5-1 µl Klenow enzyme (2-5 units), and incubate at room temp for 30 min.

Blunting 3' overhangs: Add 1 µl 2 mM all four dNTPs to your 20 µl restriction digest. Add 0.5-1 µl T4 DNA polymerase, and incubate at 37° for 5 min. T4 polymerase has a more active 3' to 5' exo activity than Klenow, and so is preferred for this reaction, but Klenow will work.

Blunting both a 3' and 5' end in the same reaction: using either Klenow alone or T4 polymerase alone or a mixture will work okay.

Blunting an end, and then cutting with another enzyme to produce another end that is sticky: after blunting the ends produced by the first enzyme, add ~80 µl 0.3 M NaOAC pH 5.2, phenol/chloroform extract, chloroform extract, add 3 volumes EtOH, put at -20° for 20 min., spin 10 minutes, wash with 70% EtOH, dry, and resuspend in the appropriate buffer for the next restriction digest. Then add the second restriction enzyme and proceed. (Note: I've tried just killing the Klenow or T4 polymerase by heating to 70° for 20 minutes, and then adding the second restriction enzyme. This hasn't given good results, though; I think Klenow in particular is somewhat thermostable).

Phosphatasing to prevent vector reclosures: After the restriction digest, add 1 µl Biolabs calf intestine alkaline phosphatase. Incubate 37° for 1 hr. (In the old days I used Boehringer phosphatase: they sell a high concentration type so that you can put in ~20 units to help phosphatase blunt and 3' overhang ends, which are more resistant to phosphatasing. In the old days we also used to change to an alkaline buffer for phosphatasing, but that appears unnecessary.)

Blunting an end, and then phosphatasing: after blunting, ~kill the Klenow or T4 pol by incubating at 70° for 20 min, and phosphatase as usual. There is a theoretical concern here that the unused free nucleotides will compete with the DNA ends for the phosphatase, but empirically it seems not to matter. If you're worried about this you could EtOH ppt. and resuspend before phosphatasing.

Cloning Taq polymerase PCR products by T/A cloning: Taq polymerase has an efficient terminal transferase activity that adds a single nucleotide to the 3' end of blunt ended duplex DNA. This terminal transferase activity greatly prefers adding A over adding C, G, or T, so most PCR products will have single 3' A overhangs. These can be cloned efficiently into a vector containing single 3' T overhangs on its ends. You could buy kits containing prepared vectors of this structure, but you can easily produce the equivalent yourself. After cutting 1 µg of any vector with a blunt cutter (e.g. Bluescript, using EcoRV) dilute the digest with 1 volume of 1X PCR buffer, add dTTP to 0.1 mM, add 2 U Taq polymerase, and incubate at 72° for 20 minutes. The Taq will add a single T to the 3' ends of the vector DNA. The vector will now self-ligate very inefficiently, and should accept Taq generated PCR products. In practice, however, ligations of such vectors with PCR fragments yield only ~10-50% clones with inserts, so it is best to use blue/white selection to identify colonies with inserts; almost all the white colonies will have an insert.

Concentration of DNA by ethanol precipitation

2.5 - 3 volumes of an ethanol/acetate solution is added to the DNA sample in a microcentrifuge tube, which is placed in an ice-water bath for at least 10 minutes. Frequently, this precipitation is performed by incubation at -20 C overnight. To recover the precipitated DNA, the tube is centrifuged, the supernatant discarded, and the DNA pellet is rinsed with a more dilute ethanol solution. After a second centrifugation, the supernatant again is discarded, and the DNA pellet is dried before resuspension in TE buffer.

Protocol

1. Add 2.5-3 volumes of 95% ethanol/0.12 M sodium acetate to the DNA sample contained in a 1.5 ml microcentrifuge tube, invert to mix, and incubate in an ice-water bath for at least 10 minutes. It is possible to place the sample at -20degC overnight at this stage.

2. Centrifuge at 12,000 rpm in a microcentrifuge (Fisher) for 15 minutes at 4 degC, decant the supernatant, and drain inverted on a paper towel.

3. Add 80% ethanol (corresponding to about two volume of the original sample), incubate at room temperature for 5-10 minutes and centrifuge again for 5 minutes, and decant and drain the tube, as above.

4 dissolve dried DNA in 10 mM Tris-HCl, pH 7.6-8.0, 0.1 mM EDTA (TE buffer).

Notes on precipitation of nucleic acids

A. General rules

Most nucleic acids may be precipitated by addition of monovalent cations and two to three volumes of cold 95% ethanol, followed by incubation at 0 to -70 degC. The DNA or RNA then may be pelleted by centrifugation at 10 to 13,000 x g. for 15 minutes at 4degC. A subsequent wash with 70% ethanol, followed by brief centrifugation, removes residual salt and moisture.

The general procedure for precipitating DNA and RNA is:

1. Add one-tenth volume of 3M NaOAc, pH 5.2* to the nucleic acid solution to be precipitated,

2. Add two volumes of cold 95% ethanol,

3. Place at -70degC for at least 30 minutes, or at -20degC overnight.

or alternatively

1. Combine 95 ml of 100% ethanol with 4 ml of 3 M NaOAc (pH 4.8) and 1ml of sterile water. Mix by inversion and store at -20degC.

2. Add 2.5 volumes of cold ethanol/acetate solution to the nucleic acid solution to be precipitated.

3. Place at at -70degC for at least 30 minutes or -20degC for two hours to overnight.

* 5M NH4OAc, pH 7.4, NaCl and LiCl may be used as alternatives to NaOAc. DNA also may be precipitated by addition of 0.6 volumes of isopropanol.

B. Oligonucleotides

Add one-tenth volume of 3M NaOAc, pH 6.5, and three volumes of cold 95% ethanol.

Place at -70degC for at least one hour.

C. RNA

Add one-tenth volume of 1M NaOAc, pH 4.5, and 2.5 volumes of cold 95% ethanol.

Precipitate large volumes at -20degC overnight.

Small volume samples may be precipitated by placing in powdered dry ice or dry ice-ethanol bath for five to 10 minutes.

D. Isobutanol concentration of DNA

DNA samples may be concentrated by extraction with isobutanol. Add slightly more than one volume of isobutanol, vortex vigorously and centrifuge to separate the phases. Discard the isobutanol (upper) phase, and extract once with water-saturated diethyl ether to remove residual isobutanol. The nucleic acid then may be ethanol precipitated as described above.

E. Notes on phenol extraction of nucleic acids

The standard and preferred way to remove proteins from nucleic acid solutions is by extraction with neutralized phenol or phenol/chloroform. Generally, samples are extracted by addition of one-half volume of neutralized (with TE buffer, pH 7.5) phenol to the sample, followed by vigorous mixing for a few seconds to form an emulsion. Following centrifugation for a few minutes, the aqueous (top) phase containing the nucleic acid is recovered and transferred to a clean tube. Residual phenol then is removed by extraction with an equal volume of water-saturated diethyl ether. Following centrifugation to separate the phases, the ether (upper) phase is discarded and the nucleic acid is ethanol precipitated as described above.

A 1:1 mixture of phenol and chloroform also is useful for the removal of protein from nucleic acid samples. Following extraction with phenol/chloroform, the sample should be extracted once with an equal volume of chloroform, and ethanol precipitated as described above.

Old way of Eluting DNA fragments from agarose

DNA fragments are eluted from low-melting temperature agarose gels using an unpublished procedure first developed by Dr. Roe. Here, the band of interest is excised with a sterile razor blade, placed in a microcentrifuge tube, frozen at -70degC, and then melted. Then, TE-saturated phenol is added to the melted gel slice, and the mixture again is frozen and then thawed. After this second thawing, the tube is centrifuged and the aqueous layer removed to a new tube. Residual phenol is removed with two ether extractions, and the DNA is concentrated by ethanol precipitation.

Protocol

1. Place excised DNA-containing agarose gel slice in a 1.5 ml microcentrifuge tube and freeze at -70degC for at least 15 minutes, or until frozen. It is possible to pause at this stage in the elution procedure and leave the gel slice frozen at -70degC.

2. Melt the slice by incubating the tube at 65degC.

3. Add one-volume of TE-saturated phenol, vortex for 30 seconds, and freeze the sample at -70degC for 15 minutes.

4. Thaw the sample, and centrifuge in a microcentrifuge at 12,000 rpm for 5 minutes at room temperature to separate the phases. The aqueous phase then is removed to a clean tube, extracted twice with equal volume ether, ethanol precipitated, and the DNA pellet is rinsed and dried.