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Questions related from Monika Jain
Strategy 1) I have prepared N- Nus and C- His(6-His) tagged fusion construct of my protein of interest in pET43.1a by using SpeI and XhoI restriction enzyme and Prescission protease recognition...
10 October 2017 3,016 3 View
For the expression of subtle Nus tag fusion protein I have optimized sonication for 5ml culture, 50 percent amplitude, 1pulse off 1pulse ON for 30 second. With repeated cycles with the gap of...
06 June 2017 9,132 10 View
How to decide how much concentration of protease inhibitor cocktail is required in buffer for protein expression ?
05 May 2017 6,848 3 View
I want to calculate total number of Rosetta cells by using OD 600 , please explain.
05 May 2017 1,734 7 View
I prepared rifampicin (150 mg/ml stock) in methanol and stored at 4 C but after some day methanol gets evaporated so it makes difficult to pipette out rifampin for next use. If i add more...
03 March 2017 8,378 5 View
During Quick change SDM F and R primers are complementary of each other when performing independent Forward and reverse primer PCR reaction (In different tubes) for quick change SDM then it is...
11 November 2016 5,732 2 View
I have performed Quick-change site directed mutagenesis by using stratagene protocol (Not kit). Mutagenesis primers were exact complementary and I have performed 1.Independent F & R...
10 October 2016 6,908 6 View
I am using NEB mutagenesis protocol (not kit) for point mutation, primers are designed back to back orientation and after PCR I want to perform PNK phosphorylation. I have thermoscientific PNK...
10 October 2016 1,662 1 View
I am interested to know whether a 1:3 molar ratio is ok with a 5.4 kb vector [5.4 kb after double digestion,(actual size of vector is 7.2 kb) ] and 600 bp insert DNA ? Is there any relation for...
10 October 2016 2,815 5 View
I amplified human gene cDNA(600 bp) with Q5 pol but after amplification I got 500 bp band with some nonspecific bands of less than 500 bp, then I performed gradient PCR and again I got 500 bp band...
10 October 2016 8,515 7 View
I performed quick change site directed mutagenesis but I am not getting mutated DNA my primers are exact complementary with each other and purification grade is desalted, not PAGE/ FPLC purified....
10 October 2016 7,986 10 View
I have isolated recombinant plasmid using all buffer solutions from GeneJet Mini prep plasmid isolation kit except resuspension solution. I used resuspension solution of promega Midi prep kit. I...
09 September 2016 4,405 1 View
I have designed primer for site directed mutagenesis to change only one amino acid in my interest of gene (which is cloned into pBluescript vector) so forward and reverse primer are complementary...
09 September 2016 575 6 View
How much amount of recombinant plasmid DNA is required for DH5 alfa competent cell's transformation.
09 September 2016 9,233 8 View
I have used Raji cells and these are suspension cells, I wish to know, Is "Raji A" (clone of Raji) a suspension or adherent cell line? From some source, I read "Raji A" was developed from...
07 July 2016 5,616 2 View
After cDNA preparation, I need to amplify this cDNA for my interest of gene so what is the concentration range (amount of cDNA in terms of either ng or ug) during PCR amplification. From some...
07 July 2016 8,004 7 View
During the blue-white screening, I am getting both blue and white colonies for ligated vector, Some colonies are light blue/ white for this when I shifted this plate from 37c to 4c so after some...
07 July 2016 2,349 1 View
I have 5 months old X-gal IPTG plates (20mg /ml x-gal and IPTG) for blue-white screening If I will use these plates now. Will it affect my result because of old plates ? Kindly suggest me.
07 July 2016 1,940 4 View
one week ago I had revived Ramos cell line in 10 cm plate by using RPMI 1640 Ramos medium. According to protocol, Ramos media should be 4500mg/L but I used 2000mg/L glucose. Next day I changed...
06 June 2016 3,204 5 View
what are the consequences if I will either TAE buffer or SDW for dilution of DNA sample ? What kind of changes can occur by using either one ?
06 June 2016 4,006 4 View
I want to change only one amino acid in my insert DNA for this I chose Quick change site-directed mutagenesis and designed primer for the same. Now I want to know another type of site-directed...
05 May 2016 8,698 4 View
In a case of quick change site-directed mutagenesis primer both primer (F) (R) are complementary to each other except one amino acid this may cause primer dimer formation. For preventing primer...
05 May 2016 3,070 11 View
My interest of insert DNA from Human I want to identify rare codons of E. coli in my insert DNA (gene coding sequence) From where I will get this information ?
05 May 2016 1,606 2 View
I know the protocol for DH5 alfa and BL 21 DE3 competent cell preparation. I wish to know is there any special step or any kind of change in the preparation of Rosett competent cell ?
05 May 2016 3,336 4 View
I want to clone AID enzyme in expression vector so please suggest me which vector will be suitable for this purpose ?. I am planning to clone AID ( Size approx 4000 bp ) by using PET expression...
04 April 2016 8,178 4 View
How to set annealing temperature (Tm for mutagenesis primer = 83C) and cycle number for quick change site directed mutagenesis? I want to change only amino acid through mutagenesis.
01 January 1970 550 1 View
