37 Questions 68 Answers 0 Followers
Questions related from Abir Chakraborty
Dear all, I have been struggling with transfection in Hs578t with my pure plasmid ( maxi prep plasmid). I transfected in a 24 well plate , changed the media after 6 hours post transfection. I...
09 September 2019 6,292 1 View
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%...
08 August 2019 3,089 3 View
I am planning to transfect Hs578T cell with pcDNA eGFP. Is it very hard to transfect? We have Extreme gene HP transfection reagent. Any advice from people if they have done that before?
07 July 2019 5,883 2 View
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...
06 June 2019 9,404 6 View
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...
05 May 2019 6,722 4 View
I have been struggling with a cloning and its due to the fact that It is digested only with a single restriction enzyme(I have no other option). I was wondering why do we need 1 pmoles of DNA for...
04 April 2019 2,514 10 View
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...
03 March 2019 4,610 3 View
Dear All, Can we incubate Supernatant and Ni charged beads for binding at room temperature for overnight? Currently, we do not have a fixed temp room in our building. What about keeping Ni...
02 February 2019 6,801 6 View
Can we store pierce glutathione agarose beads in 50 mM tris, 150 mM NaCl,pH 8.o after regeneration?
11 November 2018 7,105 3 View
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...
11 November 2018 2,482 1 View
Recently I designed a primer that is 50 bp long with 9 bp overhang.GC% is 32 and Tm 63C. I made this long primer just to get good GC%. Do you guys think I made any mistake? On this note, my...
10 October 2018 7,566 0 View
Dear All, I have digested my vector with ApaI and my PCR product is flanked by ApaI site, which I use to cut the insert from pGEM T vector after subcloning. We use CIP to prevent self ligation of...
10 October 2018 2,125 5 View
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...
10 October 2018 3,998 3 View
I have cloned a short part of ECM protein that has a signal sequence. I checked the the instability index of this protein in expasy tool with and without signal peptide. This protein is unstable...
09 September 2018 8,006 9 View
Dear All, This might be a very stupid question. Can you explain the mechanism by which Normal HEK293 cells become immortalized after being transfected with Sheared Ad type 5 DNA?
08 August 2018 2,136 2 View
Dear All, Is there any one in this group who has successfully transfected and expressed recombinant Fibronectin fragment into HeLa or HEK293 cell.
08 August 2018 4,148 1 View
Dear All, Sonication is a vital step in protein purification and over sonication can definitely damage the secondary structure of the protein(correct me if I am wrong). What is the best Hz value...
06 June 2018 9,184 6 View
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,...
05 May 2018 7,448 3 View
I have been trying to subclone an insert having 2300 bp into pGEm T easy vector. The primers I used have SnaBl and Notl site in forward and reverse primer end respectively. In my knowledge,...
03 March 2018 10,067 9 View
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 5,782 5 View
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...
01 January 2018 2,042 2 View
I need to make 1M potassium phosphate buffer pH 6.51. I have KH2Po4 and k2Hpo4,3H20. Acoording to the calculation , pH=pKa+log salt/acid I added 0.691 M KH2po4 and 0.309 M k2HPO4,3H2O for 100 ml...
12 December 2017 8,195 0 View
Hi All, I have cloned my gene of interest into pPIC9 vector following electroporation in GS115 strain. I used HIS4 gene for the selection and I got many colonies.Now, the colony should all be...
12 December 2017 5,283 5 View
Dear All, Does UV light have any role on the activity of Ampicillin? I made some Amp Plates and kept them under UV after pouring for 20 mins till it is set. Do you think I should avoid that...
12 December 2017 6,430 9 View
Dear All, I have pGem T easy vector which has T over hang. I was wondering can we insert/create T overhang in the Pgem after buying it from promega in our lab? In that case, which Restriction...
11 November 2017 2,398 2 View
I am planning to design a primer which has an overhang .This overhang has the gene of 6x His tag following RE site and extra five A's to the end of the RE site(to increase the...
10 October 2017 4,012 4 View
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...
10 October 2017 3,499 2 View
Dear all, Recently, I have been struggling to purify a protein which has 10 disulfide bonds. I have found out that the proteins are inside the inclusion bodies. I have used sarcosyl to solubilize...
09 September 2017 7,880 12 View
Dear All, Does anyone know the sequence of p-RARE plasmid or any unique restriction enzyme cut site just to confirm that the plasmid I have isolated is a right one? I have gone through few...
08 August 2017 1,478 1 View
Dear All, I have cloned my gene of interest into p-QE-80L.After that, I co transform pRARE and the plasmid with the insert into the XL1 blue. 0.25mM IPTG concentration and 24 C were used to...
08 August 2017 9,497 3 View
Dear all, I have been trying to express a protein with 10 disulfide bonds using SHuffle E.coli strain which allows the formation of disulfide bonds in the cytoplasm.MW of the protein is around 50...
08 August 2017 574 22 View
Recently I have been doing some cloning but the problem is when I do the PCR to confirm the cloning it is giving me positive results, but when I use the same construct(In which I assume it...
06 June 2017 4,618 7 View
Hi guys, This question might be a stupid one but I had some argument on it. What I understand is that bacteria can not grow in 10% SDS solution. We generally use SDS to lyse them. Am I right? Some...
06 June 2017 9,575 7 View
Lower molecular weight bands in SDS-PAGE is not properly separating. Specially 11kDA and 17kDa. what can be a cogent explanation of this issue?
02 February 2017 8,183 7 View
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 4,267 5 View
I have been struggling with BiFC complementation assay. I have pBiFC VC155 and VN173 plasmids. I cloned my gene of interest and confirmed its expression in Western blot,but I could not see any...
01 January 1970 2,680 1 View
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 3,032 3 View
