Paikwin Li

10 Questions 16 Answers 0 Followers

Questions related from Paikwin Li

When doing CRISPR KI, can I use lenti CRISPR V2 without fluorophore for gRNA delivery?

The cell line I planned to do CRISPR KI was already tagged by GFP. Two genes are going to be sequentially knocked in with mcherry and mBFP. So I planned to use pcDNA 3.1 for donor DNA and lenti...

03 November 2021 1,744 1 View

Can I use infusion cloning to insert gRNA into lentiCRISPR V2?

I intend to do CRISPR KO. Can I insert gRNA into lentiCRISPR V2 via infusion cloning? Also, will the whole sequence of lentiCRISPR V2 be incorporated into host genome or only partially? Where...

21 August 2021 7,804 2 View

Could missense mutation lead to altered expression of the gene per se? What may be the mechanisms behind this?

I'm working on a certain protein (protein A). It has been constructed on a plasmid, and two point mutations have been created (WT and mutant 1 and 2). After transfecting cells with equal amounts...

09 July 2021 1,999 10 View

What is the effect of UV on DMSO?

In my lab, DMSO is always placed on the bentch. I wonder as time goes by, will light and UV effect its stability and effectivity when used as a cryoprotectant in cell culture? Thank you for your help!

05 January 2020 4,604 0 View

When seeding cells, should I disperse them into single cells or allow them to adhere as clusters?

I'm wondering which is better for cell seeding, dispersing them into single cells or cell clusters. I thought single cells was better since cells would in this case have a more uniform growth...

24 December 2019 7,751 2 View

Cell I synchronize cells by serum starvation followed by thymidine block followed by nocodazole?

I initially plan to synchronize cells at G2/M using nocodazole (after 22 hours of serum-free media plus a two-hour recovery with complete media). However, I just learnt that it would be better to...

24 December 2019 6,542 0 View

When arrest cells with double thymidine block, how long should I arrest and release the cells?

I'm planning to synchronize Hela cells at early S phase using double thymidine block. I've read different protocols, suggesting different time periods for the incubation of cells after the...

20 December 2019 5,684 0 View

Since Hela cells do not show contact inhibition, how much does it matter that I always subculture them timely?

Since Hela cells do not show contact inhibition, how much does it matter that I always subculture them timely? Subculture cells when they are at arount 80% confluency is good practice, but can...

15 December 2019 9,815 2 View

How can I efficiently denature my double-stranded DNA sample for Urea PAGE gel?

I am doing an enzyme activity assay of an endonuclease which uses annealed oligo DNA as substrate and cleaves at one strand and releases a single-stranded DNA fragment. This strand that is gonna...

12 December 2019 225 2 View

Will frequent change of media conceal cell culture contamination?

Is it possible that under daily change of media, a contaminated cell culture may manifest no visual signs of contamination? If so, when the cells look healthy and normal, is it safe to use these...

04 December 2019 2,432 5 View