Lakshani Perera

17 Questions 18 Answers 0 Followers

Questions related from Lakshani Perera

How to clone the GOI into ROSA26 donor plasmid?

I am new to CRISPR Knock In. I want to clone a gene into ROSA 26 donor plasmid to Knock In a gene into the mouse genome. I have selected the addgene plasmid # 74285. But I cannot figure out where...

10 October 2019 9,535 1 View

Hello, I have a CRISPR related question. Can anyone help me?

I wanted to knock out MSH2 in mice cell line CH12F3 and after gRNA construction, transfection and single cell sorting, I got single colonies. I used two guide RNAs to give a deletion but when I...

05 May 2019 8,060 6 View

If I’m expressing a mammalian protein in bacteria, can I use an antibody for the protein that has species reactivity for human, for Immunobloting ?

for recombinant proteins do we have to use specific antibodies?

04 April 2019 9,779 3 View

How to design best primers to amplify a region in mice genomic DNA?

What is the database I should select under the Primer Pair specificity parameters in NCBI primer design tool?

03 March 2019 7,045 2 View

How do I amplify a 7.4 KB fragment in mouse genomic DNA ?

I am struggling to amplify an 7.4 kB fragment which I need to confirm my CRISPR KO. I have tried Pfu and Phusion polymerase, used both HF and GC buffers, tried 50 - 70 C temp gradient, DMSO...

03 March 2019 1,387 4 View

Has anyone tried using zeta probe membrane in DNA slot blotting?

I do slot blotting to my DNA samples that contain Carboxy rhodamine tags and then scan through typhoon (CY2) to get the signal intensity. Unfortunately membrane looks terrible and I cannot...

01 January 1970 2,867 0 View

Is there anyone familiar with DNA labeling using Cu-free click reaction?

I am going to label DNA bound with an azide using Cu-free click reaction. I am using DBCO-CY5. Does anyone know a suitable protocol for this?

01 January 1970 3,484 4 View

Can anyone suggest a recipe to make an Electroporation buffer to transfect CH12F3 cells ?

I’m having trouble to transfect CH12F3 cells. I tried lipofection using lipofectamine, it didn’t work. We have a biorad gene pulser xcell and I’m thinking to do an Electroporation.

01 January 1970 3,180 3 View

Anyone familiar with DBCO -CY5?

I need to purify DBCO CY5 since it give lots of unwanted bands when run on PAGE. I am looking for a protocol for that.

01 January 1970 268 5 View

Can anyone help me with this Urea-PAGE gel issue?

When I run my 17mer DNA oligos on PAGE gel I can see a smear above my DNA band. Does anyone know a possible reason or how to minimize this? I have attached a gel image here as well. Thank you

01 January 1970 905 4 View

Can anyone suggest me a method to analyse altered replication dynamics other than DNA fiber analysis?

One of the compounds that we synthesized in our lab can result in replication fork collapse. We have tested for gmma H2AX foci that shows the ds strand breaks. But we need to test for the...

01 January 1970 6,416 0 View

Can anyone help me with this question?

what is the most reasonable mechanism for hydrothiolation of terminal alkynes?

01 January 1970 7,714 0 View

Can anyone suggest me a suitable solvent system for Cl and azide separation where both are linked to n-hydroxyphthalimide with an ethyl group?

Cl and azide linked to n-hydroxyphthalimide

01 January 1970 1,822 2 View

Is the amine group of alkoxyamine visible on proton NMR in CDCl3 solvent?

Is the amine group of alkoxyamine visible on PMR in CDCl3 solvent?

01 January 1970 3,545 4 View

Has anyone used pTrac99A-rat FABP vector for protein expression ?

Is there any advantage of having Rat-FABP for protein expression using this vector?

01 January 1970 7,004 0 View

Anyone have used Zeta probe membranes?

anyone have used Zeta probe membranes?

01 January 1970 865 0 View

Are organic Azides stable in salt form ?

I am planing to synthesize an alkoxamines that contain a ethyl azide group in salt form. Does anyone know the stability of these compounds ?

01 January 1970 3,393 4 View