I am going to do for the first time and I have some basic queries. I need the help of someone who has done it.
I haven't done it, but the above idea sounds good. Exposure to intense UV light might also be worth a shot.
Simple way to induce random mutagenesis is exposure to various time points of UV ranging from 5 min to 30 min followed by phenotype screening to identify the mutants.
Hii
First of all i would suggest you to decide in wat way u want to do random mutagenesis . whether by physical or chemical means. for physical way use UV exposure dor different time interval from seconds to minutes and then screen them according to their activity
and by chemical means u can use different agaents , i have used sodium azide at different concentration and then go for screening ur mutants.
at last for mutation analysis try to go for RAPD, RFLP, AFLP or SNP analysis anyone of them i have used RAPD which will confirm whether ur mutation have occured or not.
I hope this will help you out
Dear Dash
I am attaching a protocl (Random Mutagenesis by Error-Prone PCR) which will help you step by step for better understanding and learning.
Best Regards
Dear Dash
I am also gonna attach a book for you here for more options about mutagenesis. Whish is Methods in Molecular Biology Volume 57 (In Vitro Mutagenesis Protocols).
You will learn a lot and get much expertise on different techniques about mutagenesis.
Best Regards
I have used the 'Entranceposon' system from Thermo Scientific. It lets you randomly mutagenize a target gene or genes and the nice thing (an advantage over Error Prone PCR) is that you don't get any premature stop codons. It lets you look at small coding sequence changes and in theory leaves the majority of the protein/gene in tact. I used it for a structure-function analysis in the lab and we've gotten a good amount of data from the study.
Thank you everyone for your time and useful advice I think I will go with the error prone PCR first, that I have in house. if, it doesn't then I may try some other strategy,as Michael and Afzal have recommended. thanks to all of you.
Please see the papers for your reference
https://www.researchgate.net/publication/311668087_Improvement_of_chitinase_Pachi_with_nematicidal_activities_by_random_mutagenesis
https://www.researchgate.net/publication/289367579_Characterization_and_directed_evolution_of_BliGO_a_novel_glycine_oxidase_from_Bacillus_licheniformis
https://www.researchgate.net/publication/282153514_Enhanced_nematicidal_potential_of_the_chitinase_pachi_from_Pseudomonas_aeruginosa_in_association_with_Cry21Aa
https://www.researchgate.net/publication/277326677_Improvement_of_glycine_oxidase_by_DNA_shuffling_and_site-saturation_mutagenesis_of_F247_residue
Regards
Article Improvement of chitinase Pachi with nematicidal activities b...
Article Characterization and directed evolution of BliGO, a novel gl...
Article Enhanced nematicidal potential of the chitinase pachi from P...
Article Improvement of glycine oxidase by DNA shuffling, and site-sa...
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