Abir Chakraborty

21 Questions 33 Answers 0 Followers

Questions related from Abir Chakraborty

Glutaraldehyde cross linked 0.2% Gelatin as an alternative to poly L lysine?

Hi All, Can I use 0.2% Gelatine coated, glutaraldehyde cross-linked glass coverslips as an alternative to Poly-L- lysine coated coverslips?

07 November 2020 2,331 1 View

What is the ideal Elution buffer composition for a His tag protein purification to prevent protein aggregation after freeze thaw ?

Hi All, Recently, I purified 8 proteins and stored at -20 with the elution buffer containing 20 mM Tris, 250 mM NaCl and 200 mM Imidazole. I autoclaved the buffer, so I do not know whether the...

26 September 2020 9,987 6 View

How many washes with regeneration buffer are enough for a 10 ml column containing Ni resin?

Hi All, We recently used HisPur-NiNTA resin for protein purification. The protocol from Thermo says that 20 mM MES,100 mM NaCl, pH 5 is required to regenerate beads without affecting Ni content....

15 August 2020 4,201 3 View

Max extension time for pcr with Phusion Hot start II DNA polymerase?

Hi all, I am planning to PCR a 7430 bp fragment from a big plasmid(11 kb) with Thermo Phusion hot start II DNA pol. Thermo recommend to use 15s-30s as extension time per 1 kb length. Overall GC%...

09 August 2019 7,487 3 View

How efficient and good is NEB Q5 Site Directed Mutagenesis kit?

Hello All, I just want to know your thoughts on NEB Q5 site directed Mutagenesis kit. Has anyone used this ? Is it good ? I have a plasmid of 6 kb on which I design my SDM . Your advice and...

19 June 2019 1,684 7 View

Tween 20 or Triton X in Confocal?

Dear All, Can we use Tween 20 instead of triton X in 0.1% PBT-T(I assume T stands for triton X only) for immunofluorescence ? Also, can tween 20 be replaced by tryton in IP wash buffer?

29 May 2019 6,272 3 View

What is the maximum insert length limit for ligation?

Hi All, I am planning to clone one of my inserts into VC155 BiFC plasmid. My insert size is 8 kb where vector size is 5kb. Is it very hard to clone 8 kb into 5 kb? Also I would like to do...

22 May 2019 7,107 3 View

What is the alternative way to clean Ni-NTA column other than GuHCL?

Hi All, I need to wash and recharge Ni-NTA column. Unfortunately, I do not have GuHCL now. Can we use 8M Urea instead of GuHCL? Is it also necessary to wash recharged beads with Urea+ 0.2 M...

07 March 2019 8,442 3 View

Impact of N terminal His tag on signal sequence of secretory protein?

Hi All, I recently generated few constructs in pcDNA 3.1 HisC,encoding three different Fibronectin fragments.I did not realised that His tag was at the N terminal(I just overlooked). I...

17 November 2018 5,221 1 View

Can we store pierce glutathione agarose beads in 50 mM tris, 150 mM NaCl,pH 8.o ?

Can we store pierce glutathione agarose beads in 50 mM tris, 150 mM NaCl,pH 8.o after regeneration?

01 November 2018 4,443 3 View

Alkalaine phosphatase mediated dephosporylation procedure?

Dear All, This is going to be a very silly question to ask. I have digested my vector(~4000bp) with ApaI and I need to dephosphorylate to prevent self ligation. I looked at the NEB protocol for...

09 October 2018 5,117 2 View

Recipe for making mineral salt medium?

Dear All, Does anyone have the recipe for making mineral salt medium ? I have found some information but i have few question. Mineral salt medium (MSM) had the following composition (per...

05 September 2018 333 0 View

Transient transfection and expression of Fibronectin N terminal fragments?

Dear All, Is there any one in this group who has successfully transfected and expressed recombinant Fibronectin fragment into HeLa or HEK293 cell.

09 August 2018 4,591 1 View

Blunt end and cohesive end ligation?

Dear All, I have a ~2300 bp insert with Snabl and NotI respectively( 5 prime to 3 prime). I need to use pPIC9 expression vector which is 8 kb in size. I co digested pPIC9 with SnabI and NotI,...

28 May 2018 3,738 3 View

Effect of LB broth on expression in comparison to 2x YT?

Dear All, I have been doing the expression and Purification of GST-HSP90M domain in BL21 9DE30 strain. I used to use 2x YT broth, 16 degree C induction with 0.2 mM IPTG for overnight induction....

19 March 2018 1,839 0 View

Plasmid transfer with a filter paper without cover?

Hi all, I need to take two plasmids with me to Scotland for my research work. This question seems very stupid in this group. If I add 10 ul of a plasmid on a filter paper, dry them at RT and...

03 March 2018 7,481 4 View

Effect of Ethanol on transfection in HELA?

Hi all, I had to make some endo-tox free plasmid but there is still some ethanol in the final eluted volume.I added 80 ul TE water for a total eluted volume of 200 ul.I stored them at -20 but it...

31 January 2018 2,606 2 View

Optimized protocol for Pichia pastoris?

I am planning to use Pichia pastosis for protein expression and purification. I Shall be using pPIC9 which has a secretion signal and the protein can easily be purified from the supernatant. Can...

28 October 2017 5,630 2 View

I am trying to purify HSP90 from E.coli but the problem is that Induction profile is very low. no viable band( very low once). 1mM IPTG I used?

What Should I do now? I used pet vector and BL21 (DE3) strain.

30 January 2017 5,933 4 View

Software to predict protein interface in Docked protein complex?

Hi All, I have recently used Cluspro to dock two of my proteins. I received my docked file and best model score model was named as Model000.000 something. Now, I would like to use this complex to...

01 January 1970 4,597 4 View

What is the an ideal format of writing a letter to a PI for a post doc position?

Dear All, It would be very helpful if some of you who are PI in Universities and Research institute and receive a bunch of postdoc application every month, share your opinion and suggestion with...

01 January 1970 9,851 5 View