Molecular Biology Lab
Molecular Biology Lab BIO 181
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This 2 page Class Notes was uploaded by Clarissa Hermiston DVM on Monday October 5, 2015. The Class Notes belongs to BIO 181 at California State University - Sacramento taught by Amy Rogers in Fall. Since its upload, it has received 18 views. For similar materials see /class/218814/bio-181-california-state-university-sacramento in Biological Sciences at California State University - Sacramento.
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Date Created: 10/05/15
Some notes for labs 7 10 Understand and be able to fill out a table of possible ligation products like we did for lab 7 This includes predicted which products of ligation would be propagated requires an origin of replication and which would confer antibiotic resistance Cloning strategies handout ex 3 Be able to determine appropriate restriction sites that give a desired orientation after ligation of the insert DNA into a vector In this case EcoRI XbaI or KpnI XbaI but NOT EcoRI KpnIConsider additional factors 1 N E Compatibility of chosen enzymes for simultaneous double digestion While it is possible to do one digest first purify the DNA and then perform the second digestion this is not desirable It is best if the two enzymes can work in the same buffer and at the same temperature If one enzyme is slightly less efficient this can be compensated for by increasing the of units 0fenzyme used or increasing the time of digestion Proximity of the two cut sites in the multicloning site If they are very close digestion with one enzyme may leave the second enzyme s recognition site at the end of the DNA molecule Some restriction enzymes will not bind and cut near the ends of DNA some will You would need to check this out Always minimize the amount of junk DNA in your construct Choose restriction enzymes that cut as closely as possible to the exact region you wish to clone This is particularly important if you want to express a gene when extra DNA can cause problems with transcription or translation Transformation results 9 plates Expected growth patterns A K L amp plates kan plates ampkan Satellite colonies on amp not kan plates ampicillin resistance is mediated by an enzyme 5 lactamase that degrades the antibiotic This creates a locally reduced concentration of antibiotic around each resistant colony Kanamycin resistance is NOT mediated by the destruction of the antibiotic so no zone of lower concentration exists even around resistant colonies Furthermore kanamycin is bactericidal not bacteriostatic For transformations of the ligation Most colonies on amp fewer on kan fewest on ampkan plates Why 1 All products of the ligation that we discussed in lab 7 are equally likely to form in the ligation reaction However once transformed into bacteria more of these products can confer ampicillin resistance than kanamycin resistance and only one can confer resistance to both antibiotics Also with kanamycin a long recovery period is crucial for survival of transformed cells due to immediate lethal effect If the recovery period is somewhat abbreviated one expects to see more colonies on the more forgiving ampicillin Calculate transformation efficiency for pAMP amp pKAN only Lab 9 Replica plating sample data below From Ligation plated on amp 1 10 0 no growth Ligation plated on kan 1120 Satellite colonies from Amp plates shouldn t grow anywhere Lab 10 Interpretation of gels the products of pAMPpKAN ligation 0 First determine which of the four fragments are present there must be at least 2 0 Second look at bands of UNCUT miniprep DNA and compare sizes with UNCUT pAMP or pKAN This will help you determine whether the plasmid has 2 or 4 fragments Use these data to determine whether 1 or 2 plasmid types are present and which DNA fragments they are made of Importance of double transformants bacteria can be transformed by more than one plasmid Cloning in general Understand and be able to describe the steps in cloning a genepiece of DNA into a plasmid vector We did all of these in labs 710 1 2 4 gt1 Plan a cloning strategy This protocol will assume two different sticky ends are used Digest both vector and the source of the insert DNA with the same two restriction endonucleases If possible do these as double digests both enzymes at the same time Confirm that complete digestion has occurred Run an aliquot of each sample on a gel to look for evidence of partial digestion If partial digestion is found add more enzyme and or continue digesting for more time This is to prevent having intact plasmid in the transformations as intact plasmid transforms at a much higher efficiency than ligated plasmid confounding your ability to get the recombinant plasmid you want M the restriction enzymes so they do not compete with DNA ligase Ligate Mix your cut vector and cut insert DNA together Add DNA ligase and buffer containing ATP amp Mg Incubate at room temperature to encourage annealing of sticky ends which is diminished at higher temperatures Transform Add your ligated DNA to competent cells prepared from midlog phase broth cultures Depending on the antibiotics to be used for selection of transformed cells allow an adequate recovery period for expression of the antibiotic resistance genes on the plasmid vector Screen Observe and interpret the growth patterns on your plates Select colonies from your desired selection plate in our experiment Ligated DNA onto AmpKan Grow some of them in broth cultures Isolate plasmid DNA by miniprep method Perform appropriate restriction digests on miniprepped plasmids to characterize which clone colony carries the recombinant plasmid you want
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