Glen Drake

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Questions related from Glen Drake

Centrifugation of peptide nanoparticles cause aggregation or increased particle size?

I want to use a RALA peptide vector, and I have found that some studies centrifuge the plasmid/RALA complexes and resuspend the pellet before use, while others seem not to do so. It seems like the...

06 June 2023 2,561 2 View

Kozak sequences for genes without a second codon that starts with G? Or, kozak sequence when +4 G is impossible?

The Kozak sequence is 5'-gccRccAUGG-3', but the only amino acids that can be coded for by codons that start with G are valine, alanine, aspartic acid, glutamic acid, and glycine, so how is...

09 March 2023 8,252 1 View

Identification of endogenous terminator sequence?

I'm working with a non-model organism, and have recently come to understand that different terminator sequences have significant impact on RNA localization and translation efficiency, so I want to...

23 February 2023 3,270 0 View

Single cell holder/plate/well for secreted bioluminescence imaging?

I want to use secreted bioluminescence to identify successfully transfected cells, however that would require each individual cell is in its own little isolated well so that the successfully...

22 February 2023 7,671 1 View

Most mild promoters for expression of rtTA to avoid metabolic strain on organism? Or, least toxic transactivator?

I'm trying to make a stable transgenic line of a rather delicate eukaryote, so I want to activate transgene expression inducibly, but that means I need to express some kind of intermediary protein...

07 February 2023 7,469 0 View

How necessary is it to have a G after the start codon for efficient translation?

I have been under the impression a Kozak is just GCCACC preceding the start codon, but just learned it also includes a G just after the start codon, i.e. GCCACC ATG G. If I am encoding a peptide...

06 February 2023 6,545 0 View

Non-toxic reporter gene or other transgenic DNA integration verification method that does not kill/damage organism?

I have been thinking that I will just use EGFP to know whether my attempted transfection was successful, but I've been coming across some publications that say over expression of GFP can cause...

22 January 2023 4,290 2 View

Explanation for benchling's gibson assembly tutorial homology arm primers? Homology arm concatenation?

Benchling's tutorial for automatic generation of primers for gibson assembly, https://help.benchling.com/hc/en-us/articles/9684273545997-Clone-using-the-Gibson-Assembly-Wizard , states that "Note:...

17 January 2023 6,930 0 View

Order oligos for CRISPR gRNA synthesis, or make them yourself?

I'm new to using CRISPR, and all the protocols I'm reading are just telling me to order the oligos for the primers for gRNA synthesis. Is this what everyone does, or is there a way to feasibly...

15 January 2023 7,734 3 View

PEI delivery of RNP + plasmid?

Can I use PEI to simultaneously deliver a CRISPR RNP and the plasmid? Obviously PEI can package and deliver the plasmid, but I am having trouble finding information about PEI's ability to package...

11 January 2023 5,733 1 View

100 nm diameter or less transfection vector for large plasmid or 5k bp plasmid + RNP?

I have an idea for an organism i want to make, but to produce them i will likely need a vector that is no more than about 100 nm in diameter. I have been considering using BSA-PEI particles, but i...

11 January 2023 352 0 View

Monoavidin/streptavidin for binding large(6-7k bp) biotinylated donor DNA?

I found this study, https://www.biorxiv.org/content/10.1101/248179v3.full, and I really want to use the technique, but I'm concerned that monoavidin may have trouble binding a large DNA fragment...

18 September 2022 7,324 0 View

Size limit on a CRISPR monoavidin-biotin-donor DNA link?

I'm looking at this study, https://www.biorxiv.org/content/10.1101/248179v3.full , and it uses a monoavidin - biotinylated donor DNA link to create a fusion of the donor DNA and the RNP so that...

18 September 2022 6,639 0 View

Can lipofectamine be used for generation of large gene(6-7k bp) knock-in thru CRISPR mediated HDR?

This was my original plan, however I was just reading this review A review of emerging physical transfection methods for CRISP... and it states that "When using lipofection, the cargo only...

14 September 2022 5,134 1 View

Best membrane for quantitative western blot of complex II?

Hi, I'm trying to measure the relative abundances of complex II of the ETC with a western blot. I know PVDF is best for low abundance proteins and nitrocellulose is best for low background noise,...

04 April 2019 4,051 4 View

Negative Microarray Log2 Value?

Hi! I'm looking at this study Mice Fed Rapamycin Have an Increase in Lifespan Associated w... , and their excel microarray data has negative log2 values, which doesn't make sense to me. Can...

04 April 2019 5,373 4 View

Monoavidin - biotinylated DNA binding size limit?

Is there a limit on the size of DNA fragment that can be bound by a biotin-monoavidin bond?

01 January 1970 498 1 View