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Title: what are steps for inserting a gene into a plasmid from a cell of interest? Post by: smag on Sep 17, 2012 i knwo you have to use the restriction enzyme and taek it out of that but hwo do i place it together?
Title: what are steps for inserting a gene into a plasmid from a cell of interest? Post by: mightypulpo on Sep 17, 2012 The cut out gene and the bacterial plasmid are mixed 2gether in a test tube and since they've been cut with the same restriction enzyme, they have identical "sticky ends", so the palsmid recombines with the gene spliced into it. the splicing 2gether of the plasmid with the gene inserted into it is catalysed by the enzyme DNA ligase.
The plasmid is re-inserted into the bacteria by mixing with calcium chloride in a test tube. Title: what are steps for inserting a gene into a plasmid from a cell of interest? Post by: miharu on Sep 17, 2012 Here are the 7 steps for gene cloning using plasmids....
STEP 1. A gene of interest ( DNA fragment) is isolated from cells that have been grown in lab. culture ( you perhaps prepared it in the laboratory, have not you?); STEP 2. Both the human DNA and the plasmid are treated with the same RESTRICTION ENZYME to produce identical sticky ends; STEP 3. The restriction enzyme cuts the plasmid DNAat it's single recognitin sequence, disrupting the TETRACYCLINE resistance gene; STEP 4. The DNA fragment are mixed together and the complementary sticky ends are attached to each otherby base pairing. The enzyme DNA- LIGASE is added to bond the sticky ends; STEP 5. The recombitant plasmid , or molecular clone, is introduced into a bacterial cell by adding the DNA to a bacterial culture. Under the right condition some bacteria will take upthe plasmid from solution by the process TRANFORMATION; THIS IS THE PREPARATION OF CLONE upto this point. NOW, let's start CLONNING the gene...... STEP 6. The actual gene cloning process ( making multiple copies of human gene ) occurs when the bacterium with the recombinant plasmid is allowed to reproduce; STEP 7. Colonies of bacteria that carry the recombinant plasmid can be identified by the fact that they are resistant to ampicillin but sensitive to tetracycline. That's all. Are you happy of the answer? Or did you expect to know something more. E-mail me, then only I can help you. Best Wishes. |