분자 생물학 DNA , RNA 추출 리포트

분자생물학 (14102946 김동윤) 1학기 실험 보고서.hwp

Title: Plasmid DNA Extraction by QIAprep Spin Miniprep Kit and Electrophoresis

 

준비물 : 1.5ml tube, DNA marker, Buffer P1,P2,N3,PB,PE,EB,TAE

Loading dye, EtBr, Agarose, QIAprep Spin Miniprep Kit

사용 장비 : Pipette, 전기영동기구 , Microcentrifuge , UV Lamp

Used Bacteria : Plasmid　DNA　Cloned Vector Transformed E.coli (BL21)

 

Step 1 (Bacteria pDNA Extraction)

 

01. pellet 1ml bacteria overnight culture by centrifugation at > 5000 rpm for 5 min

at room temperature 15-25＇c and Allow the culture to flow

02. resuspend pelleted bactria cells in 250 μl Buffer P1(lysis buffer) abd transfer to a microcentrifuge tube.

03. add 250μl buffer p2(SDS) and mix thoroughly by inverting the tube 10 times.

until the solution be comes clear Do not allow the lysis reaction to proceed for more than 5 min.

04. add 350μl buffer N3(Acetic acid) and mix immediately and thoroughly by inverting the tube 10 time.

After the previous experiment 1 min standing in room temperature.

05. centrifuge for 10 min at 12,000 rpm in a table-top microcentrifuge.

06. apply the supernatant from step 5 to the Qiaprep spin column by deconting by decanting or pipetting Centrifuge 60s and discard the flow-through

07. wash the QIAprep spin column by adding 500 buffer PB centrifuge for 60s and Should be discarded the flow-through

08. wash the QIAprep spin column by adding 750μl buffer PE. centrifuge for 60s Transfer the QIAprep spin column to the collection tube and Should be discarded the flow-thorough.

09. centrifuge for 1 min to remove residual wash buffer

10. place the QIAprep column in a clean 1.5ml microcentrifuge tube. to elute DNA,

add 500μl buffer EB(10m tris-cl , ph8.5) let it stand for 1 min, and centrifuge for 1 min

 

Step 2 (Electrophoresis)

 

01. <Agarose gel production> 200ml TAE Buffer + Agarose 2g Mix Stir well until dissolved -> + EtBr 15ul

02. Mix the pDNA sample and Loading dye in a 1.5ml tube at a 3: 1 magnification (sample 15ul, dye 4ul)

03. dispensing It will harden after about 45 minutes.

03. Fill the electrophoresis buffer with TAE buffer and add agarose gel.

04. Pipet the marker into the prepared sample on agarose gel.

05. Perform electrophoresis for about 20 minutes.

06. After electrophoresis is complete,take out  the gel and check the result with UV lamp.

 

Step.3 (Experiment result)

 

01. Overall, the shape of the marker is not accurate, but it shows a fine shape.

02. Markers 3 and 4 have a distinctive shape, which is presumed to be well distributed.

03. Although the pDNA seems to be present, the result of the pDNA that we were trying to confirm

did not perform enough electrophoresis and the marker size could not be measured.

04. Since the sharpness of the marker is low in the agarose gel, it is judged that there is a technical error.

05. I think I need to practice repeatedly because I lack pipetting skills.