4 What is lambda(λ)? Why lambda? Lambda is a bacteriophage, virus that infects bacteriaInserts its nucleic acid into the host bacterial cellReplicates rapidly inside host cells until the cells burst and release more phagesHarmless to man and othereukaryotic organismsSOOOO, excellent source of DNA for experimental study.
5 Lambda genome is about 48,000 bp If linear lambda DNA is cut with HindIII, how many fragments will there be? Longest piece? Shortest piece?
6 What is a restriction enzyme? Enzymes that cut DNA at specific places known as restriction sitesAlso called endonucleasesBacteria use them as a natural defense against bacteriophagesBiotechnology – cutting genes from one organism and pasting them into another – would not be possible without these enzymes
7 Enzyme Site Recognition Restriction sitePalindrone• Each enzyme digests (cuts) DNA at a specific sequence = restriction site• Enzymes recognize 4- or 6- base pair, palindromic sequences(eg GAATTC)Fragment 2Fragment 1
8 5 vs 3 Prime Overhang • Generates 5 prime overhang Enzyme cuts• Generates 5 prime overhangDNA from any organism cut with the same enzyme will produce complementary sticky endsWhen mixed together, complementary bases will hydrogen bondLigase is needed to reform the phosphodiester bonds.
10 How is a restriction digest done? Restriction Buffer provides optimal conditions for enzymeWhy incubate at 37°C?Body temperature is optimal for these and most other enzymesWhat happens if the temperature is too hot or cool?Too hot = enzyme may be denaturedToo cool = enzyme activity lowered, requiring longer digestion time
11 How can we separate all those fragments of DNA? Agarose gel electrophoresisAgarose is purified agarDerived from seaweedagar provides a medium on which bacteria (and other microorganisms can grow)agarose provides a “sieve” for separating DNA fragments by sizeLarge fragments travel slower than small fragments
12 “swimming through Jello” Gel electrophoresisDNA is negatively chargedwhen it’s in an electrical field it moves toward the positive sideDNA –+“swimming through Jello”
13 Agarose Electrophoresis Loading • Electrical current carries negatively-charged DNA through gel towards positive (red) electrodeBufferDyesAgarose gelPower Supply
14 How can we see the DNA fragments – DNA is not colored? Stain them!The stain we use is relatively nontoxic, easy to handle and dispose ofBUT it’s not very sensitive so in “real life” other types of stains are usedLoading dyes and tracking dyes do not stain DNA; they just help you see where your sample is and how far DNA fragments have probably traveled
15 How can we determine the sizes of the DNA fragments? Run a standard in one of the wells; also called marker or ladderStandard has been cut with restriction enzymes and the size in base pairs (bp) has been determinedCompare migration of fragments whose size is known to the migration of fragments whose size is unknown
16 How can we determine the sizes of the DNA fragments? Create a standard curve using the migration of the marker DNA fragmentsDetermine the size of the unknown fragments from this graphSemi-log graph paper will be needed
17 Analysis of Stained Gel Determinerestriction fragmentsizesCreate standard curve using DNA markerMeasure distance traveled by restriction fragmentsDetermine size of DNA fragmentsIdentify the related samples
18 Molecular Weight Determination Fingerprinting Standard Curve: Semi-logSize (bp) Distance (mm)23,000* 11.09,6,4,2,2,*This fragment falls outside the linear portion of the curve. You may choose to exclude it from your best fit line
20 LESSON 1Page 24 Let’s answer the two questions in the middle of the pageBe sure to fill out the chartPage 25 You can answer – 2 questions at the top and the 4 review questions at the bottomPage 26 You can answer – 4 questionsPage 27 You can answer – 4 questions
21 LESSON 2 Page 30 You can answer – 5 questions Page 32 At the top – Let’s answer that now
22 LESSON 3 follow procedure 2. a on page 35 Page 36 Do not attach your tracing here; hand in separately; if you have no data another group will share with you – be sure to document where the data came fromPage 37; if you followed directions the wells are in this order:Lane 1 Marker (we know the size of these fragments; theywere cut with HindIII )Lane 2 Uncut lambdaLane 3 lambda cut with PstILane 4 lambda cut with EcoRILane 5 lambda cut with HindIII
23 LESSON 3 Measure from the front of the well to the front of the band RECORD DATA ON PAGE 38BE CAREFUL GEL IS FRAGILE!
24 LESSON 3 Page 39 You do – 6 questions Page 40 You do – 2 questions Clarification step 3 use markerPage 42 – Complete the graph – you may ignore the 23,000 bp piece when drawing the best fit linePage 43 in the “estimated” columns also include the data from the first gel analysis in parenthesis; the number from the standard curve graph should not be in parenthesisPage 44 You do – 4 questions
25 Lane 1: marker, lambda cut with HindIII Lane 2: uncut lambdaLane 3: lambda cut with PstILane 4: lambda cut with EcoRILane 5: lambda cut with HindIII
26 Lane 1: marker, lambda cut with HindIII; 7 sites, 8 pieces Lane 2: uncut lambda; 48,502 bpLane 3: lambda cut with PstI; 28 sites, 29 fragmentsLane 4: lambda cut with EcoRI; 5 sites, 6 fragmentsLane 5: lambda cut with HindIII
27 WHY DIDN’T WE SEE ALL THE FRAGMENTS AS BANDS IN THE GEL? SOME BANDS ARE SO CLOSE IN SIZE THEY DID NOT SEPARATE USING THIS PROTOCOLSOME FRAGMENTS ARE SO SMALL THEY CAN NOT BE DETECTEDHow could we get better results?Change gel concentrationLonger run timeMore sensitive DNA stain
28 EXPERIMENTAL ERROR*Incorrect measurement*Not using optimal temperature for enzyme for the right amount of time*Some of sample did not enter well*Stock solutions not kept on ice