Q1: What is the format requirement of the input sequence?
A1: The format of input sequence should be in plain text format. To make it convenient for users to deal with the sequence, the software is designed to ignore case and punctuation of the input sequence.
Q2: What is the [Monovalent cations]?
A2: It is the total concentration of monovalent cations in the PCR system, including Na+, K+, Tris+, NH4+, etc. We provide a default value of “50mM”, and users should change it according to the actual composition in the reaction mixture prior to run.
Q3: What is the difference in designing the one-site SDM primers and multi-site SDM primers?
A3: It just needs to do one round of PCR for one-site SDM, while the multi-site SDM needs two. Tm of the “OL” region of each pair of multi-site SDM primers is designed higher than one-site SDM primers, up to 58-62°C in the software, so the neighbor fragments can be jointed in the second round of PCR,
Q4: How to choose one-site mode or multi mode?
A4: It is determined by whether more than one pair of primers is to be designed at the same time. The one-site mode means not only to introduce just single nucleotide mutation, but also multiple mutations spanning a fragment longer than 50bp. In other words, the distance between mutation sites determines the number of primers to be designed. For the distance between the neighbor sites longer than 50bp, it is necessary to choose multi-mode to design more than one pair of primers to do mutations at different sites.
Q5: How to design the overlap extension primers for DNA fragment assembling with this software?
A5: To assemble several DNA fragments is similar to do multi-site SDM. On the webpage of multi-mode, the input box should be filled with the connected target sequence. The number and nucleotide of each junction should be filled in “mutate code” box in sequence, with the format like “C30” (30th “C” is the last nucleotide of previous sequence).
Q6: How to design primers for vector construction?
A6: There is a special module to do primer design for vector construction. The process is similar to do DNA fragment assembling. The flanking sequences of vector and target gene should be input in the left box of the “Vector-construction” module, and the number and character of the connection nucleotide should be filled in the right box. It is convenient to run separately to generate two pairs of primers for junctions of vector and gene. To be note, the junction nucleotides should be shifted to the vector, making the vector primers the same for different target genes, so that there should be a pair of universal primers for each vector.
Q7: What are the PCR conditions to do mutations with primers from PS?
A7: For one site mutagenesis, we suggest a standard 20μl PCR reaction system containing about 10ng template plasmid, 0.5U KOD DNA polymerase (Toyobo), 0.2mM each dNTPs, 2μl 10×KOD reaction buffer, 2mM MgSO4, and 0.25μM each mutagenesis primer. The PCR program is as follows: 94°C 2 min for initial denaturation, and start the 18 amplification cycles for 94°C 25 s, 55°C 25 s, 68°C for “N” min (determined by the plasmid length and 1kb per min for KOD), at last with a final extension step for 10 min at 68°C.
For the multiple-site mutagenesis, it is recommended to do two rounds of PCR. The first-round is to separately amplify the fragment between two neighbor mutation sites. The PCR mixture is similar to one site mutagenesis，to run with each forward primer and neighbor rewards primer. It needs to amplify more cycles to get a higher yield of fragments with mutations. After gel purification to remove template plasmid, the second round of PCR is to run with fragments mixed at equal molar ratio and the two farthest apart primer pairs.
Connect with us:
For general questions in using, please contact with Xianhui Hou
(Email: email@example.com )
For questions in primer design processing, please contact with Zhiyong Pei