Sayanti Halder

13 Questions 12 Answers 0 Followers

Questions related from Sayanti Halder

Should we stirred T4 DNA polymerase in the vial before using?

We are using pET-32 Xa/LIC kit (Novagen 70072-3) for cloning. T4 DNA polymerase given with the kit to generate compatible overhangs on the insert. After T4 DNA polymerase treatment we we have...

23 August 2021 5,586 7 View

Is there any difference between NovaBlue GigaSingles™ Competent Cells and DH5α competent cells (Hi media) for transformation?

We are using pET-32 Xa/LIC kit (Novagen 70072-3) for cloning. NovaBlue GigaSingles™ Competent Cells given with the kit are not viable, so we have used DH5α competent cells (Hi media) for...

13 August 2021 1,990 4 View

Ideally is there any difference between LB or SOC media to select transformants after cloning?

We are using pET-32 Xa/LIC kit (Novagen 70072-3) for cloning. In kit manual there is no media mentioned for plating the transformants. We have used the LB culture medium. But there is no colony...

13 August 2021 4,376 4 View

Is there any way for sequencing of our PCR purified target aptamer except cloning?

Our target aptamer sequence is small (78bp). We are facing a problem during cloning. Is cloning necessary for sequencing of our target aptamer (DNA) or any other way is present? Any suggestions...

05 August 2021 1,089 3 View

What may be the reason behind formation of lawn instead of colony, on LB plate of positive control insert during Ligation independent cloning?

We have transformed positive test control insert (given with pET-32 Xa/LIC cloning kit) with E. coli DH5 alpha competent cells. After 24hours of incubation on LB plate (40 µg/ml ampicillin...

04 August 2021 2,298 5 View

Anyone help me with size of test plasmid of pET-32 Xa/LIC cloning kit?

We have transformed the test plasmid of pET-32 Xa/LIC cloning kit with E. coli DH5 alpha competent cells. After 24hours of incubation on LB medium (50 µg/ml ampicillin containing medium) at 37°C,...

21 July 2021 1,619 4 View

Facing problem during recombination and transformation using pET-32 Xa/LIC cloning kit?

Our desired insert is small (around 78 bp long). We have used 0.02 pmol (as per kit protocol)of desired insert for annealing with pET Xa/LIC Vector. Then we transform 1 µl annealing reaction...

19 July 2021 7,794 2 View

How we can calculate number of molecules of aptamer into molarity?

I find in different articles that for first round of SELEX they have used different number of aptamer molecules (like 1014, 105 etc). We have 70µM aptamer stock. Whether is there any formula to...

14 May 2021 7,953 1 View

What should be the optimum aptamer-protein ratio during first round of SELEX?

We have 24 amino acid in our peptide sequence. 24 amino acid- (24×110) = 2640g/mol =Molecular weight. Molecular weight of out aptamer- 24,009.5 We are taking 6.46 µl of protein from stock. (116...

14 May 2021 7,067 2 View

What should be the optimum final working concentration of aptamer for aptamer-SELEX procedure?

We have 68.9 µM of aptamer stock concentration. During SELEX procedure we have taken protein coated Ni-NTA beads & we have taken 7.25 µl aptamer having concentration of 68.9 µM, then diluted...

13 May 2021 1,983 0 View

During aptamer selection process (SELEX), whether aptamer-protein conjugate interfere during PCR cycle?

I'm trying to quantify aptamer (received from SELEX round) by PCR. As after elution we got protein-aptamer complex, is it necessary to remove that protein from aptamer-protein complex & use only...

01 May 2021 9,568 1 View

Can anybody suggest how to remove eluting buffer(2% SDS) from SS DNA aptamer solution to be used in second round of SELEX?

We are using Streptavidin Mag Sepharose kit for eluting SS aptamer DNA, in elution buffer 2% SDS is present. We are planning to remove this SDS through gel extraction kit. Is any other suitable...

01 May 2021 752 1 View

Is it possible to use binding buffer instead of Tris buffer, for eluting SS aptamer DNA using gel extraction kit?

We have single stranded aptamer DNA, we want to elute that aptamer by gel extraction kit (Sigma). This SS DNA (aptamer) could be eluted on the basis of pH? We have binding buffer (20mM Sodium...

01 May 2021 2,166 0 View