Tips and Useful Information for DNA Cloning

Epicentre’s Scientists frequently need to clone and express genes as part of their R&D projects. These genes may be cloned directly from restriction enzyme-digested genomic DNA or cDNA or, if the gene sequence is known, the insert may be obtained by PCR amplification using genomic DNA or cDNA as a template. Whatever the source of the insert, they believe the following tips will enable you to efficiently and successfully clone your gene.

Tip # 1. Don’t expose your insert to UV light.
UV transilluminators have been used for many years for visualizing DNA bands separated on agarose electrophoresis gels. Damage to DNA by the UV light is enough to be detectable after even 5 seconds of exposure and enough to make certain regions unreadable on a sequencing gel within 60 seconds. One solution to avoid exposure of the gel-separated DNA to UV is to run an extra duplicate lane next to the lane with the fragment to be cloned. The duplicate lane is cut out of the gel, stained and visualized on the transilluminator and then used as a marker for where to cut out the band from the lane that is not exposed to UV. A better solution is to use a Dark Reader™ Visible Light Transilluminator from Clare Chemical Company. A Dark Reader uses only visible light, which does not damage DNA and is not harmful to people, and two filters. The gel is first exposed to light that has passed through a blue filter. It filters out all wavelengths except the excitation wavelength of the intercalator dye. Then, the bands are viewed through an orange filter, which only allows the excitation wavelengths to pass through. Sensitivity using SYBR Gold or other dyes exceeds UV light with ethidium bromide. More important, exposure to Dark Reader light does not affect cloning efficiency. In one experiment at Epicentre, a 30-second exposure of phage T7 DNA to UV light on a transilluminator decreased the number of transformants obtained using that DNA as an insert by more than two orders of magnitude, but a 5-minute exposure to Dark Reader light had no effect on cloning efficiency. See www.clarechemical.com for more information.

Tip # 2. Minimize losses of your insert DNA.
If you purify your insert DNA on an agarose gel, you will obtain quantitative recoveries by using a low-melting-point agarose gel and then digesting the gel slice containing insert band using GELase™ Agarose Gel-Digesting Preparation. Alternatively, if you’re making the insert by PCR, you can probably skip the gel and remove the nucleotides and primers by precipitating the PCR product using the DNA Fragment 2X Precipitation Solution.

Tip # 3. If you generate an insert by RT-PCR, don’t take chances by using the wrong PCR enzyme or conditions.
Avoid any PCR enzyme or reaction that uses manganese cations. For example, our MasterAmp™ RT-PCR Kit for High Sensitivity is optimized for and delivers the claimed highest detection sensitivity, but it uses manganese cations, which makes it error-prone. You should not use it to generate an insert for cloning because it is highly likely that the product will contain at least one mutation. To amplify RNA, use the MasterAmp™ High Fidelity RT-PCR Kit instead. It uses an enzyme blend with proofreading activity to obtain a PCR fidelity that is several times higher than Taq. To amplify a DNA fragment for cloning, the FailSafe™ PCR System uses a unique enzyme blend and specially optimized PCR PreMixes that permit amplification of even the most difficult templates with extremely high specificity and sensitivity, and with a fidelity several-fold better than Taq.

Tip # 4. Repair your ends.
Double-stranded DNA inserts obtained by PCR often have 3´-non-template-encoded nucleotides or otherwise imperfect ends for cloning. Special T-vectors can be used for cloning such PCR products, but we obtain even better results by blunt-end cloning after repairing the ends of the insert DNA. T4 DNA polymerase is excellent for this purpose because it has a 3´-to-5´ exo activity in addition to the polymerase fill-in activity, so the ends are perfect. This is followed by treatment with T4 polynucleotide kinase to make sure that all of the 5´-ends are phosphorylated and clonable. Alternatively, incompatible or damaged DNA ends can be easily blunt-ended and 5´-phosphorylated in a single reaction with the End-It™ DNA End-Repair Kit, which contains both T4 enzymes optimized for this use.

Tip # 5. Increase insert cloning efficiency by eliminating vector self-ligation.
Treatment of the vector with a DNA phosphatase will prevent self-ligation, but permit ligation of the 3´-ends of the vector to 5´-phosphorylated ends of the insert DNA. The nicks on the unligated strands are then repaired in the host cell following transformation. Epicentre introduced the first thermolabile DNA phosphatase (HK™ Phosphatase, from an Antarctic bacterium) in 1988, but it is no longer the best solution for cloning.

Tip # 6. Use a good ligase.
Our Technical Services Scientists have heard about many instances in which the ligase made the difference between success and failure in a cloning experiment. Ligases vary in quality. At Epicentre, we go to great lengths to make sure our ligases are the highest possible quality, and that makes a difference. We and our customers have had good success with the Fast-Link™ DNA Ligation Kit, which is optimized for high-efficiency room-temperature ligations in 5 to 15 minutes, depending on the type of DNA ends being ligated. Don’t try to save 5 more minutes here. Use the right amount of time that is optimal for the type of end you have. For optimum transformation efficiency, the DNA ligase in the reaction should be heat inactivated at 70°C for 15 minutes before transforming the competent cells. In some cases, it is also beneficial to ethanol precipitate the ligated DNA in order to reduce salt content for electroporation.

Tip # 7. Use competent cells with an optimal genotype for your cloning purpose.
Competent cells should be chosen based on their transformation efficiency and their ability to support specific vector features. All of Epicentre’s competent cells retard degradation of plasmid DNA (endA 1 minus); are defective in recombination, so plasmids are maintained as monomers (recA 1); allow efficient cloning of methylated and nonmethylated DNA (restriction minus); and can be used for blue/white screening (lacZΔM15). For library construction, they also accept large DNA fragments without size bias up to at least 23 kb, to greater than 145 kb, depending upon the cells. Please see our competent cells selection guide.

Tip # 8. A kit may be the best option.
Some people think they will save money by assembling all of the individual reagents themselves. To be honest, it’s probably less expensive (and easier) to buy a kit, such as the CopyControl™ cDNA, Gene & PCR Cloning Kit. It contains a linearized and dephosphorylated vector, DNA Fragment Precipitation Solution, end-repair reagents, Fast-Link Ligation reagents, DNA size markers, competent cells, lysis reagents, and controls, all of which are optimized and pre-tested. Even more extensive kits are available for construction of complete BAC, fosmid, or cosmid libraries. There are also three different kits for screening recombinants based on insert size, PCR, or restriction analysis.

Happy Cloning!

The Genetic Code
1st Position (5´)	2nd Position				3rd Position (3´)
	U	C	A	G
U	UUU Phe UUC Phe UUA Leu UUG Leu	UCU Ser UCC Ser UCA Ser UCG Ser	UAU Tyr UAC Tyr UAA Stop UAG Stop	UGU Cys UGC Cys UGA Stop UGG Trp	U C A G
C	CUU Leu CUC Leu CUA Leu CUG Leu	CCU Pro CCC Pro CCA Pro CCG Pro	CAU His CAC His CAA Gln CAG Gln	CGU Arg CGC Arg CGA Arg CGG Arg	U C A G
A	AUU Ile AUC Ile AUA Ile AUG Met	ACU Thr ACC Thr ACA Thr ACG Thr	AAU Asn AAC Asn AAA Lys AAG Lys	AGU Ser AGC Ser AGA Arg AGG Arg	U C A G
G	GUU Val GUC Val GUA Val GUG Val	GCU Ala GCC Ala GCA Ala GCG Ala	GAU Asp GAC Asp GAA Glu GAG Glu	GGU Gly GGC Gly GGA Gly GGG Gly	U C A G
AUG initiation codon is in bold green. GUG codes for Met if in the initiation position. Stop codons are in red.

 

Amino Acid (AA) Symbols and Properties
Amino Acid	3-Letter Symbol	1-Letter Symbol	Molecular Weight		Charge	Hydro- Phob.**
			Free AA	In Peptide
Alanine	Ala	A	89	71	0	0.31
Arginine	Arg	R	174	156	+1	-1.01
Asparagine	Asn	N	132	114	0	0.60
Aspartic Acid	Asp	D	133	115	-1	-0.77
Asparagine/Aspartic Acid	Asx	B	–	–	–	–
Cysteine	Cys	C	121	103	0	1.54
Glutamine	Gln	Q	146	128	0	-0.22
Glutamic Acid	Glu	E	147	129	-1	-0.64
Glutamine/Glutamic Acid	Glx	Z	–	–	–	–
Glycine	Gly	G	75	57	0	0.00
Histidine	His	H	155	137	+1	0.13
Isoleucine	Ile	U	131	113	0	1.80
Leucine	Leu	L	131	113	+1	1.70
Lysine	Lys	K	146	128	0	-0.99
Methionine	Met	M	149	131	0	1.23
Phenylalanine	Phe	F	165	147	0	1.79
Proline	Pro	P	115	97	0	0.72
Serine	Ser	S	105	87	0	-0.04
Threonine	Thr	T	119	101	0	0.26
Tryptophan	Trp	W	204	186	0	2.25
Tyrosine	Tyr	Y	181	163	0	0.96
Valine	Val	V	117	99	0	1.22
* Also add +1 for an N-terminal free amine and -1 for a C-terminal free carboxyl group. ** Calculate hydrophobicity for a peptide or protein as described in Fauchere & Pliska. (1983) Eur. J. Med. Chem. 10, 369.