Having trouble cloning?

Plasmid construction is constantly going on in nearly all molecular biology labs. Although nobody would like to describe cloning a piece of DNA into a vector as a major obstacle to a research plan in a grant application, or a glorious achievement in a publication; cloning could be, and often is the most time-consuming and mind-boggling step in a project. A typical theme in DNA construct creation starts with preparing a vector by restriction enzyme digest and insert DNA by either digest or PCR. The two pieces are then ligated together before transforming into competent bacterial cells where the ligated DNA molecules are amplified and selected.

The key to a successful execution of this procedure relies on retrieving correct DNA fragments before ligation. DNA isolation and recovery are currently done with PCR/gel extraction kit that utilize a silicon membrane immobilized inside a column, which can bind DNA (e.g. from a PCR reaction or a band cut out of a gel) in the presence of guanidinium. While this is a common practice in biological experiments, something often thought to be quite simple and straightforward; in reality it sometimes takes weeks or even months, repeat after repeat, before successful cloning is achieved. To increase cloning efficiency, people turn to “Super” competent cells, high concentration ligase, automated colony pickers, or high throughput sequencing for help.

Many sub-cloning projects get stuck due to plasmid recombination, by which a piece of DNA rearranges into a smaller plasmid than intended, often a bare-bone minimum plasmid that includes only the replication origin and antibiotic-resistance gene. This problem is amplified when either or both the vector and the insert fragments are large, or contain repeat sequences that destabilize DNA, such as those on viral vectors. Low efficiency of cloning is also a significant problem during library construction where a high degree of diversity is required. Recombination is facilitated by DNA nicks or breaks, something that can result from UV damage during gel viewing or by harsh chemical reagents in current DNA purification kits. The following is a recent real case of sub-cloning experienced by Allele Biotech researchers in our San Diego molecular biology lab:

Objective: cloning a group of 5 cDNAs (different versions or fragments from one gene transcript) into a retrovirus transfer plasmid for viral packaging
Vector: pCHAC1, ~12 kb, with terminal repeats
Insert: ~0.4-1.7 kb
Using standard PCR/gel purification kits (Allele Biotech), dozens or hundreds of colonies were obtained in each of the 5 rounds attempted, all of which were incorrect with various sizes below the projected size, including bare-bone (~3kb) plasmids. Different competent cells, (e.g. chemically competent DH5a, electro-competent DH10b), secondary structure-tolerant strains, etc. were tried to no avail.

Changes: Avoid all UV exposure and harsh chemical reagents, use solid surface binding that tethers DNA after each restriction digest or PCR directly in the coated PCR tube in the presence of a special binding buffer, and elute DNA into just the required volume of reaction buffer for the next reaction, e.g. ligation, transformation.

Results: 4 out of 5 constructs were made after only one round, with more than half of the colonies examined being correct. The failure of the 5th one was attributed to an orientation mistake in the parental plasmid used as PCR template.

Conclusion: DNA damage during gel running, cutting, and DNA extraction can severely hinder the creation of a difficult DNA construct.

New Product of the Week: magnetic beads-based surface-bind DNA purification kits, email oligo@allelebiotech.com for details.

Promotion of the week: Promotion of the week: 10% off all Media (Insect Media, FBS, Cell Selection Media and more). To redeem this offer email abbashussain@allelebiotech.com with promo code Media10

Tags: cloning difficulties, colony screening, competent cells, DNA cloning, DNA purification, DNA recombination, ligation, molecular cloning, plasmid construct, surface bind

New SurfaceBind gDNA Isolation and Purification

Allele Biotech’s SurfaceBind Genomic DNA Pu¬rification Kit is designed for fast, easy, and high-throughput gDNA isolation and purification for lysate obtained through the use of Allele-in-One Mouse Tail Direct Lysis Buffer. Based on our Solid Surface Revers¬ible Binding (SSRB) technology the SurfaceBind system utilizes a plastic tube with its surface coated with proprietary turbo-binders acting to selectively capture and efficiently bind DNA mol¬ecules from reaction mixtures. After lysis of cells, gDNA molecules will specifically interact with the turbo binders and bind to the surface of the tube in the presence of the binding buffer, while pro¬teins and other contaminants will remain in solu¬tion. The DNA can be eluted with as little as 10 microliters of water or buffer for the next application, allowing for a highly concentrated solution.

The entire process of recovery takes less than 10 minutes with only 1 centrifugation step, making it fast and easy. SSRB technology also provides for maxi¬mum DNA capture and release with limited sam¬ple input, without the DNA loss associated with membrane and bead-based technologies.

This is a newly developed product particularly for the Allele Biotech’s customers who use the All-in-One mouse tail genotyping kits: get purified genomic DNA using the same lysate you generated for a quick PCR. The yield and purity will enable direct applications to chip assays, sequencing, Southern blotting, etc.Next time you use Allele-in-One Mouse Tail Direct Lysis Buffer be sure to try our SurfaceBind gDNA Purification kit.

mWasabi-GFP Expression vector with IRES for co-expression. Cat # ABP-FP-WIRES10. email FP@allelebiotech.com for more FP IRES-containing plasmids.

10% off all mTFP1-expressing plasmids this week, check out the vectror you like at shop.allelebiotech.com

Tags: DNA purification, genomic DNA, microarray DNA, mouse tail genotyping, next generation sequencing, one step PCR, Southern Blot

Purifying DNA without Membrane Binding

Purifying DNA from natural samples or biochemical reactions is one the most frequently performed experiments in virtually all molecular biology labs. Binding DNA to silica membranes in chaotropic binding buffers is the currently prevailing method, pioneered by Qiagen. Before Qiagen columns and the similar columns from a number of companies, including Allele, there was the silica resin, mostly from Promega. Before silica, it was phenol extraction or CsCl gradient.

Silica-based technology has been around for more than a decade, and it is time for a new generation of technologies that are more convenient and efficient than silica membrane to take center stage of DNA purification, especially given the fast-paced advances in polynucleotide analysis in microarrays and deep sequencing. Solid Surface Reversible Binding (SSRB) technology should be a shining star in coming years. The process is simple: DNA or RNA molecules in a simple binding buffer bind to the surface of plastics of any size and shape (PCR tube, 2.0ml eppendorf, 96-well plates, even 15 ml or 50 ml conical tubes) that is treated by a special process, washed, and eluted in any volume of water or even downstream reaction buffers. The utmost convenience is that the downstream reaction can be performed in the same tube!

This process is different from the electroreversion type of binding and releasing that requires buffers of different and extreme pH. Allele Biotech has started marketing SurfaceBind PCR purification kits, and will roll out products that are specifically tailored for genomic DNA, mRNA, size-differentiated DNA or RNA, DNA or RNA from fixed samples, from different species, etc. The convenience and cost-efficiency of these systems will provide significant contributions to the broad scientific community.

New Product of the Week 101810-102410:

SurfaceBind PCR purification kit, questions? Please email us at oligo@allelebiotech.com for a product description and introduction quotes.

Promotion of the Week 101810-102410:

Updated: GFP-nAb products (equivalent to previously distributed GFP-trap), good for genomic DNA pull down by transcription factor-GFP fusion, RNA co-IP via RNA binding protein-GFP fusion. Or try our brand new, the brightest mFP–mNeonGreen and already available mNeonGreen-nAb, or anti-mNeonGreen nano antibody (also referred to by others as “nanobodies”).

Tags: deep sequencing, DNA purification, genomic DNA, microarrey DNA sample, miniprep, miRNA, nextgen sequencing, PCR cleanup, PFA linked, RNA purification, silica membrane, surface resersible binding