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 forward primer has just 36 base pairs(GC% 44 and T m 65C ).
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...
08 September 2019 6,186 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%...
07 August 2019 2,980 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?
06 July 2019 5,737 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...
05 June 2019 9,287 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...
04 May 2019 6,586 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...
03 April 2019 2,416 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...
02 March 2019 4,485 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...
01 February 2019 6,671 6 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...
10 November 2018 2,354 1 View
Can we store pierce glutathione agarose beads in 50 mM tris, 150 mM NaCl,pH 8.o after regeneration?
10 November 2018 6,963 3 View
I have a dataset with about 80 different species. As usual, some species are very easy to identify with certainty whereas others are more difficult, which means that I am less certain of my...
03 March 2021 8,066 4 View
Hello, We would like to increase the yield of our PCR product. We are running a series of PCR reactions that is targeting ~1.1kb sequence. We begin each reaction with ~400pg of template DNA...
02 March 2021 4,029 3 View
Hi, I am planning to apply for the PhD degree in the Supply Chain Mgt. with specific area of "Cold Storage warehouses" during Pandemics and wars. Where lock downs and shut downs are frequent....
02 March 2021 285 2 View
So, I have been trying to run a pACYC PCR which will be used later on for a Gibson Assembly. However the PCR is not working. I have already tried gradient PCR and changing extension time; however...
02 March 2021 1,146 2 View
I have to amplify a gene and my primers just reached. The Tm for Forward primer is 64.2, and that of reverse primer is 65.5. Can some one suggest how to get the best annealing temperature? Thanks...
01 March 2021 360 7 View
To dear Researchers, I was analyzing a series of concentration for estimation of Real-Time PCR efficiency. The concentration was 1:10. I used MS-excel to evaluate Slope. The result of slope was -8...
01 March 2021 8,683 4 View
I am trying to identify these 3 genes among some tomato cultivar collections and after aligning some sequences from NCBI, I couldn't find unique sequences to target for specific primers. There...
28 February 2021 606 3 View
hello everyone, I need to do standard curves for my qPCR, what is the ideal efficiency range? I tried a primer (Mglu2 receptor) that gave an efficiency of 90.2%. Is it accepted?
28 February 2021 1,254 3 View
Dear All, mirna primer showing some problem in the melting curve? any idea why? As attached is the melting curve. The forward sequence is obtained from miRBase and reverse primer is universal.
28 February 2021 5,008 4 View
I performed site directed mutagenesis, transformation, and then I sent out plasmids for Sanger sequencing and found out that there is extension of DNA just before the stop codon. I am not sure...
27 February 2021 547 3 View