University:
Swansea University
Course:
PM134 | Microbiology
Academic year:

2023
Views:

445

Pages:

3
Author:

Sanjeev KG

ISOLATION OF DEGRADATIVE PLASMIDS IN MICROBES GROWING IN POLLUTED ENVIRONMENT AIM To isdate degeadat we plasmids in microbes growing in Polluted environments. PRINCIPLE Farially these processes do seperation on the basis 2 physical differences between cheornosomal DNA 4 Plasmids ike their ige , which is ene malt obvious difference The size of largest plasmid of Ecoli is only 8% of the cheomosomal DINA. Therefore techniques can seperate aut the large DOVA molecules them small diged DNA casily used to purity plasmids. can be Further moll also disser chromosomal DNA of bacteria af plasmids with respect to theer conformation it means wrey have different special corfanations of the ndicelles. As plasmids have circular momosomal DNA has lincar conformation but due to breaking down ducing the process of are janed the irumlar plasmids are being und onbaction usually + the proutses chrom of Orzes lineas fragments. that can Hperate from the einces brun fragments. the plasmid of degradative property was isdated by selective wolation of by supplementing the compound in the media. MATERIALS REQUIRED Bujza Pi: 50MM Teil NCI, PH-80 10mMEOTA, 100mg/ml Einst A store of 42), Buyer Dam Noon; 1% (w/) 5275 Buffer Pa PROCEDUCE Foulteral welate from which degradation placmid. Hay to be Holated was allowed to gras in a minimal media supplemented with a degradative ingard which lect wacth of selective. roulated then the walleiss Incletin single sceny medin scallowed wat interdigd CANTS to grow the 12-10 he to obtain the peelet I centrifuge acttuls at maximum sperrt of PT for imin 2. completely scaneve superlintant Restuspend 12 35041 bufjes. Pi Add 350 el buffer Pa for upto 5 min at RT. Gently miss by inverting incubate 4 Add 400ml buffer P3. Gently miz by inverting. (ide not 5 E Centrifuge too lomins at Et af maximum speed. Carefully thanger supernatant silts, contaminatios to a teeth ture, with of pellet material al potible necessary antipye the supernatant again to completely. seman pellet isopropanol precipitation 2 And I volume 2 min of isopropanol Incubat On Gatlinge sample dos soming at RT el maximum speed cacefully remove mpernatant without discarding the AVA pellel 7. wash pellet by adding sooull") 70% eenonel samples for Smins al RI al mar speed. ney Pellet ing thermoblock al 65°C tos semin 1. Rakussines pellet in 50-100 ml bure water & heppe of chall ( Te hyjee) OBSERVATION Ourge coloured bands were observed this indicates the Juence of plasmid ONA. RESULT The degradative plasmids were successfully isolated from missobes gowing in polluted environment.