Lab Exercise DNA Extraction

10/30/13 DNA in all organisms is found primarily in chromosomes. Eukaryotic cells, like those found in humans, contain several linear chromosomes. Prokaryotic cells, such as bacteria, contain one large circular chromosome. DNA is composed of building blocks called nucleotides, each made up of a five carbon sugar !deo"yribose#, a phosphate group, and a nitrogenous base. $he four kinds of nucleotides differ only in their nitrogenous bases. %t is the nucleotides and their bonding pattern that give DNA its uni&ue characteristics and functions. 'tructurally, DNA contains t(o long strands of bonded nucleotides. $hese strands run in opposite directions. $he t(o long strands are connected by hydrogen bonding bet(een the nitrogen bases of the nucleotides !A $ and ) *#. +ach bonded pair of nucleotides is called a base pair. An average gene consists of a se&uence of appro"imately 3000 base pairs. $he functional units of DNA, the genes, code for polypeptides (hich serve countless functions for the cell. ,ithout proteins !for e"ample, en-ymes#, the chemical reactions that make up a cell.s metabolism could not proceed in a timely manner and the cell (ould die. /acterial chromosomes contain appro"imately 3000 genes. 0umans, on the other hand, contain about 10,000 12,000 genes in each of their cells. %f all of the DNA in all of the cells of a human (ere stretched end to end, it (ould form a ladder 100 billion kilometers !you could climb to the sun and back 300 times#. $oday, you (ill e"tract DNA from the bacterium Micrococcus luteus. $he first step is to lyse !burst# the M. luteus cells to release the DNA into the solution. %n order to do this, a detergent used in laundry products called 'D' !sodium dodecyl sulfate# is used to degrade lipids in the bacterial cell membrane. ,hen the cell membrane is damaged, the cell lyses, releasing the cytoplasm.s contents into the solution. $his causes the solution to become viscous !thick# due to the long strands of DNA that are no longer as tightly coiled as they (ere in the cell. Along (ith DNA, en-ymes and other proteins are released. /ecause en-ymes harmful to free DNA must be destroyed, the solution is heated !proteins denature or lose their functional shape at 30 320)#. DNA also denatures (hen heated but only (hen the temperature reaches 400). )old ethanol, an alcohol, (ill then be added. DNA is insoluble in alcohol and (ill precipitate !come out of solution# (hen it comes in contact (ith ethanol. A spooling rod is used to remove the DNA. /y slo(ly rotating the rod in the solution, the DNA (ill attach and coil around it. Because DNA is long and thin, it can be mechanically broken quite easily. Be extremely gentle at all time.

Materials: per group of 4 1 tube of free-e dried M. luteus bacterial suspension solution !share bottle (ith other groups# 'D' !sodium dodecyl sulfate# 1ml pipet and pipet pump t(o 2ml pipet and pipet pump 1 spooling rod !glass rod# 1 large test tube containing 10ml of 526 ethanol, refrigerated test tube rack hot (ater bath !30 320 )# Procedure: 1. 1. 3. 8. 2. 3. 9. 4. 5. 7ipet 2ml bacterial suspension medium into the test tube containing free-e dried M. luteus Gently rotate the test tube bet(een your palms for appro"imately 2 minutes until the bacteria go into suspension. 7ipet 1ml 'D' into the suspension. Again gently rotate the tube bet(een your palms several times over a 2 minute period. 7lace the tube in a 30 320) (ater bath for 30 minutes. After 30 minutes in the (ater bath, remove tube and allo( to cool to room temperature. )arefully lo(er the spooling rod !glass rod# into the tube. $ip tube at 820 angle. :eep holding the tube at this angle until step 10. 0ave a lab partner pipet 2ml of cold ethanol into the tube. 0old the pipet against the inside (all of the tube !still tipped at 820 angle# and allo( the ethanol to flo( ery slo!ly do(n the side of the tube onto the top of the M. luteus solution. Do not shake the tube. "lo!ly rotate the spooling rod in a continuous clock(ise motion (hile continuing to hold the tube at an angle. Avoid touching the sides of the tube. ;ibers of DNA (ill come out of solution and attach to the glass rod like thread on a spool. )ontinue rotating the rod for several minutes until a mass of DNA is attached. 11. <emove the glass rod and gently immerse it in the tube containing the remaining ethanol for 10 minutes.

10.

DNA Extraction Lab #e$ort

1. !1pt# =ist the three components of a nucleotide.>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>> >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>> 1. !.2pt# ?n average, ho( many genes are in a bacterial chromosome@>>>>>>>>>>>>>>>> 3. !1pt# %n the e"periment you (ill add the detergent 'D' to the bacterial cells. ,hat is the purpose of adding 'D'@>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>> >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>> > >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>> > 8. !1pt# ,hen e"tracting DNA, ho( do you remove the effects of harmful en-ymes that degrade DNA@>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>> >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>> > 2. !1pt# ,hat is the purpose of using ethanol is the e"periment@>>>>>>>>>>>>>>>>>>>>> >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>> > 3. !.2pt# Describe the appearance of the e"tracted DNA on the spooling rod. .>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>> >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>> > >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>> >