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

Making primers

We often hear that oligos (primers) are a simple product, and they should work every single time. From a producer’s point of view, oligos are arguably the most complicated products among common molecular biology reagents, at least in terms of the number of chemical steps required. DNA synthesis starts from the 3’ end to the 5’ end (opposite to DNA polymerases) on a solid support (e.g. CPG beads). For the addition of each base, the process begins by removing a protection group via “Deblocking”; then activating the last base for coupling with an “Activator”, adding the current nucleotide in the chemical form of a phosphoramidite (4 times in our protocol), blocking un-reacted openings with “Capping A and B solutions” (again 4 times each), forming bonds between bases by oxidization with Iodine, then looping all the way back to the beginning of the cycle. Many of these chemicals are either highly sensitive to moisture or have a short shelf life (can go bad any time).

Coming back from a seminar, sitting at the lab desk, you know you have a new idea and some cloning to do, and of course, it must be done tomorrow. The first thing you do is to send in some oligo sequences online to a local synthesis company late in the afternoon after looking at some maps and sequences. Around noon the next day, the oligos will be delivered in person to your hands. Most times everything will just work out fine as far as experiments involving primers are concerned; others you get stuck here and there along the way of cloning. Chances are you have run into problems with primers not giving PCR signals or clones with mistakes in the primer regions at some points in your research career. Even if this has only happened a few times, the memory, as well as the dissatisfaction and anger, can last for quite some time.

Between ordering and delivering, oligos are made overnight; they are then post-synthesis processed (requiring several hours starting in the early morning), OD’ed, and concentration adjusted. Given that the machine completes the run without any problems (power or computer related), and none of the chemicals run out, the best quick indicator of a good run is a color change from the protection group removed by Deblock at the last base. If there is visible amount of blocking group at the end of the synthesis, as reflected by an orange color from a Trityl group, it is likely that the synthesis was efficient till the end. Unfortunately, the efficiency of adding each base is never 100%–accumulations of missing or, at a lower percentage, mistaken bases will add up, especially in long oligos. Purification will remove some of the oligos with deletions, but not all of the bad oligos. MASS analysis will help determine the approximate percentage of bad oligos, but it will require time and cost not typically chosen by customers. It is our hope that understanding how oligos are made will help with more effective use of oligos when you order oligos, conduct experiments using oligos, or clone with oligos.

New Product of the Week: Phosphate-3′-CPG for oligo synthesis, email oligo@allelebiotech.com for details.

Promotion of the week: Promotion of the week: 10% off on 4-in-1 lentiviral particles for iPSCs generation. Email oligo@allelebiotech.com with promocode: VIRUS, or using online purchase.

Tags: amidite, amino oligos, biotin oligos, CPGs, FAM amidite, fast turnaround, insert, oligo, oligo chemicals suppliers, PAGE, PCR, primer, probe, siRNA, trityl

The NIH is one step closer to be short of $260 million from its 2011 budget

The National Institutes of Health (NIH) 2011 budget will be cut by $260 million in the budget that the House has just passed based on the last minute pact reached last Friday to avoid federal government shut down.

The NIH’s 2011 budget will be $30.7 billion, down 0.8% from its 2010 budget of $30.9 billion, according to news releases that can be found from various sources. Previously, the President proposed a $32.1 billion budget for the NIH and the House of Representatives allocated $29.4 billion to the agency. President Obama asked for a $1 billion increase for the NIH in 2012, which will be in new debate to start immediately. Chances are the 2012 budget for the agency will be less than what the administration wanted.

Combined with “the cliff effect” from the ending of the stimulus money the NIH has epically managed since 2009 to fund extra research projects, the negative growth of the NIH budget could mean less academic positions and tighter lab budgets ahead. Cutting-edge technology and cost effectiveness will be the key for survival of the fittest in the biomedical research jungle.

Promotion of the week: Save 10% on any purchase of feeder cells. Email brianahasey@allelebiotech.com with offer code : FDST11

New Product of the week: Damage-free cloning kit for difficult cloning projects—get recombined plasmids or failed ligation? Your DNA is damaged by purification bugger and/or UV, ask us how to deal with it oligo@allelebiotech.com

Tags: 2011 budget, budget, congressional republicans, federal budget, NIH, President Obama, research budget