5 tips for working with unstable ORF expression clones

Mammalian expression constructs for certain genes such as human c-KIT are notorious for undergoing frequent recombinations during cloning and transformation steps in molecular biology labs. Experts suggest that certain genes are “toxic” to bacteria thus leading to a situation in which recombined plasmids are favored. While the molecular mechanisms for this toxicity may be unclear, the end result is that efforts to amplify, subclone, mutate, or make derivative vectors often result in a new plasmid with unwanted sequence errors.

Online science discussion forums such as ResearchGate include a variety of strategies proposed by researchers experiencing this kind of plasmid instability. Suggestions include advice such as culturing the bacteria at room temperature rather than 37°C, culturing on plates rather than in flasks, using low copy number EPI400 competent cells, picking the small colonies rather then the large ones from the LB plate, replacing the ampicillin resistance cassette to prevent satellite colonies (so you can see the small colonies), or using a Gateway vector.

But which methods work best?

[Read more…]

CleanTag small RNA library kit with no Gel Purification

 

Next generation sequencing has quickly become the preferred method over tiling arrays for most genomics and transcriptomics needs. The major exception has been the study of microRNAs, where highly sensitive probe arrays such as the 3D-Gene® miRNA profiling platform are still widely used. A large part of the reason for the persistence of array dominance in small RNA expression profiling is caused by the variability introduced in sequencing library prep protocols involving complicated hands-on PAGE purification steps.

The CleanTag™ Ligation Kit for Small RNA Library Preparation now allows users to remove the Gel Purification steps from their protocols and shift to more automated bead purification protocols. This is particularly important for cases when RNA quantity is limiting. Traditional small RNA library prep protocols will result in the formation of adapter dimers (similar to primer dimers) when RNA quantities are limiting, thus greatly reducing the number of usable reads. [Read more…]

tebu-bio collaborates with experts to find a replacement for TAP

As detailed in our previous post, the discontinuation of the enzyme tobacco acid pyrophosphatase (TAP), has left many molecular biology researchers without a suitable enzyme for their protocols. Perhaps the most classic assay using this enzyme was 5′ RACE, which required TAP to remove the 5′ mRNA cap yielding a 5′-monophosphate RNA. [Read more…]

Group purchase model for oligonucleotides in Europe

Any researcher who has purchased a complex custom oligonucleotide with chemically-modified bases has probably noticed that prices drop dramatically with increased production scale. Also, companies are reluctant to promise a final yield and prefer, rather, to issue quotes for initial starting scales.

For example, a request for custom synthesis of the modified preadenylated linker oligo below with a 5′ Adenylate and a 2′,3′-Dideoxycytidine blocking group to prevent self ligation might yield quotes for 1.0 umole starting synthesis scale (1 OD guaranteed yield) or 15 times the amount (15 OD guaranteed yield) for only about 4 times the cost.

Preadenylated linker oligo: 5′- (rApp) AGA TCG GAA GAG CGG TTC AG (ddC) -3′

In the case of this particular oligo, the 5′ Adenylate group is useful for protocols that involve the ligation to the 3′ end of RNA libraries using T4 RNA Ligase Deletion Mutant 2, however 5′ adenylated oligos require an extra purification after the adenylation step and are not compatible with heat-sensitive modifications. Consequently, PAGE purification is required leading to increased costs, particularly for small production scales.

Structure of 5′ Adenylate:

O-0264

But what if researchers could get together and order a bulk purchase of commonly used oligos and benefit from the relatively low costs of bulk production?

For the oligonucleotide above, we tested this idea by contacting multiple RNA biology groups throughout Europe performing a technique called iCLIP (individual-nucleotide resolution Cross-Linking and ImmunoPrecipitation). Developed by Jernej Ule, the next generation sequencing-based technique is used to identify specific RNA sequences bound by RNA binding proteins. Overall, the response was positive and the researchers liked the idea of sharing a single, high quality oligonucleotide production. Many indicated that the oligonucleotide called L3 seemed unnecessarily expensive due to the custom production. They also liked the idea that one researcher could quality control the oligonucleotide and others would have access to the same pre-validated production lot. We therefore launched production and created a new catalog product 3′ iCLIP Primer, and the first users have used this new product with positive results. User data provided by Jernej Ule’s former student Zhen Wang (Laboratory of Hervé Le Hir, Institut de Biologie de l’École Normale Supérieure) demonstrates that the oligonucleotide will perform well in iCLIP protocols:

New L3 test upper

Figure 1. 15% TBE-Urea gel of RNAs ligated with L3 linkers. A synthetic RNA of 40nt was used, and the ligation was performed with T4 RNA Ligase at room temperature for 2.5 h. Different PEG conditions were tested for ligation efficiency, and the old L3 linker was used as a control. In each reaction, 10pmol of RNA and 20pmol of linker was used in a total of 20ul. Gel visualized with a SYBR®-based stain such as GreenView™ Plus DNA Gel Stain.

European scientists interested in custom production of complex oligonucleotides can contact tebu-bio using this form.

Let us know if you think that your oligonucleotide has the potential to be our next catalog product!

New!:  We’ve also just released TruSeq™ compatible Index Primers 1-12 and 13-24 including RT and Forward PCR Primer for our sequencing customers, intended for use with the new CleanTag™ Ligation Kit for Small RNA Library Prep.

 

Legal notes: SYBR® is a registered trademark of Life Technologies. TruSeq™ is a trademark of Illumina.

New: CleanTag Ligation Kit for Small RNA Library Prep

As detailed in a previous post, chemically-modifying oligonucleotide adapters is an effective means to prevent adapter dimer formation during small RNA library prep. Just as primer dimers form when very little template DNA is used for PCR, adapter dimers can form with low starting concentrations of RNA. The CleanTag™ Ligation Kit for Small RNA Library Prep is a complete kit which is compatible with Illumina® technology that makes use of such modified adapters in optimized buffer conditions. The kit includes CleanTag™ chemically modified adapters that greatly reduce adapter dimer formation and is optimized for total RNA input from 1-1000 ng. [Read more…]

Expired patents might lower prices for SuperScript® II RT Users

When the patents on the SuperScript® II Reverse Transcriptase enzyme expired, a number of companies introduced their own version of the popular RT kit for cDNA synthesis. The advantage of these enzymes over traditional Moloney Murine Leukemia Virus Reverse Transcriptase (M-MuLV) is that they have been engineered to have greatly reduced RNAse H activity. RNAse H itself can be a useful enzyme for molecular biology as it specifically degrades RNA in a RNA:DNA hybrid, but it is clear to see how RNAse activity in an RT reaction is not ideal. In general the companies releasing alternatives to SuperScript® II RT aligned their pricing to be a bit less expensive then the popular Life Technologies kit. The strong brand loyalty molecular biology customers have, as we described in a previous post, meant that customers would continue to pay a higher price for SuperScript® II Reverse Transcriptase. [Read more…]

A look inside the new Otogenetics NGS library prep kits

More than a month ago we wrote a post about the new DNASeq library prep kits from Otogenetics, a U.S. based sequencing service provider who created their own kits to reduce costs. Now, we’ve had a chance to see what the kits and their contents look like… [Read more…]

Next gen chromosome analysis with dGH and Pinpoint FISH probes

dGH_Normal_and_InvertedThe U.S.-based company KromaTiD, Inc. offers a a large collection of catalog and custom chromosomal imaging paints, probes, and kits based on their patented technology. KromaTiD appears to have the only method capable of detecting so-called cryptic inversions where relatively small segments of the genome have changed orientation. Other technologies have a lower limit of inversion detection of 10 megabases, however KromaTiD’s directional Genomic Hybridization (dGH) technology allows visualization of inversions as small as 6 kilobases.

KromaTiD’s dGH kits can be directly integrated into any lab performing traditional chromosome analyses and fluorescence in situ hybridization (FISH). The assay is performed on living cells, which undergo one replication cycle in the presence of a DNA analog. The photolabile analog daughter DNA strand can then be destroyed by light leaving only the parent strand, thus allowing directional chromatid-specific imaging with KromaTiD’s probes. [Read more…]

Genome Editing in Stem Cells: outsource or do-it-yourself?

Many researchers are facing a dilemma: they want to set up a CRISPR genome editing project but they can’t decide which cell line to use for genome editing. Even some of the most cost-effective genome editing cell line generation services like the one from GeneCopoeia will cost a few thousand euros, so picking the correct cell line and setting up the project correctly is very important. Researchers working in primary cells may find the idea of switching to an immortalized cell line a bit artificial. They dream of the possibility of editing the genome of a stem cell line which they can then differentiate into their tissue of choice as needed. [Read more…]

tebu-bio supports post-transcriptional gene regulation course

tebu-bio is pleased to announce their sponsorship of the 1st Course on “Post-transcriptional gene regulation: mechanisms at the heart of networks” (Institut Curie, March 23-27, 2015) that will be training 40 PhD students and postdoctoral fellows from 10 countries. Post-transcriptional gene regulation refers to every level of gene-expression control in a cell that occurs between transcription and the resulting protein levels. Specialists in this field study such things as polyadenylation, miRNAs, RNA binding proteins, signal transduction, and (pre)mRNA splicing. [Read more…]