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Lecture 7

Lecture 7.docx

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Department
Biochemistry
Course Code
Biochemistry 2280A
Professor
Christopher Brandl

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Description
Expressing YFP in E.Coli  Overall Steps: 1. Determine which tissues express YFG (hybridization, northern blot) 2. Isolate mRNA 3. Synthesize cDNA 4. PCR YFG from cDNA 5. Gel purify PCR product of YFG (gel electrophoresis) 6. Restriction cut TFG 7. Ligase 8. Transform into E.Coli 9. Verify clones of YFG (restriction mapping, DNAsequencing) DNA Ligase ● DNAligase - glue for DNA ● Will reseal compatible sticky ends; the ends anneal weakly through hybridization ● Ligase will recognize the ends, and in the presence ofATP will reseal the ends by forming covalent bonds between them ● Ligase requires a 5’phosphates to stick the DNAs back together ● Can also perform the same process with blunt ends, but is more difficult as the sticky ends start off with some attraction to each other already due to weak hybridization Steps in Cloning ● Digest ~100ng of YFG and a plasmid vector with the same restriction enzyme or one that gives compatible overhanging sequences ○ most convenient when using the same enzyme ● After plasmid has been cut, you purify the DNAby running an electrophoresis and checking to see if it was done correctly ● Next, incubate the plasmid and insert YFG in the presence of DNAligase andATP ○ the sticky ends anneal (hybridize) and ligase seals the ends with covalent phosphodiester bonds ● This results in a recombinant plasmid ● How do we get this recombinant plasmid into the bacteria (E.coli) now? Transformation ● The process by which cells take up DNAfrom their environment ● Natural property of some bacteria; E.coli must be treated with chemicals to do it ● Effectively, all you do is you mix E.coli with ligated DNA, leave them be for ~2 minutes and then some of the E.coli will have taken up the plasmid ○ the efficiency is not very good but it is usually more than enough required ● Now that E.coli has the recombinant DNAinside, you take the transformed cells and place them on agar-plates containing antibiotic ○ the antibiotic only selects for the cells containing the plasmid; others will not and will die to the antibiotic ● Cells with the plasmid will divide forming visible colonies on the plates ● Note, not all of the transformed bacteria contain a plasmid that has YFG inserted. Reasons for this include: ○ Ampicillin (antibiotic) resistant colonies may appear (unlikely) ○ The plasmid vector can circularize without YFG ■ there’s a good chance that when you add ligase, the ends that were originally cut, may simply reform without the YFG even attaching ■ to avoid this, either add a lot of YFG or remove the phosphates off the plasmid (ligase requires phosphates to bind) ○ Contaminating DNAmay be ligated into the vector Checking the Clones ● Amplify bacteria in individual colonies ● Isolate the plasmid DNA ● Verify which “clones” contain YFG ○ restriction map (most common method) ○ hybridization ○ PCR ○ sequence Restriction Mapping ● ADNAmolecule can be defined by the number and positions of its restriction enzyme cut sites ● Because you know how far apart the certain cut sites are, and you know which restriction enzymes you’re using, you will know how many pieces the plasmid will be cut into, and how long (in base pairs) each section is ● Then run a gel electrophoresis and you can compare the predicted restriction map with the actual map and if they match, you’re cloning has been done correctly ● To be sure that YFG is correct, it should be sequenced as only sequencing will tell you exactly what those sections of cut plasmid are DNA Sequencing  Materials required to sequence a DNA ● DNAto be sequences (template) ● Oligonucleotide primer they anneals to the template ● DNApolymerase ● dNTPS (G, A, T, C) ● ddNTPs (small amount, 0.2%) Process ● To figure out the sequence, we use small amounts of each nitrogenous base (A,T,C,G) that are defective and add in polymerase to the mix of plasmid portions ○ normal DNAnitrogenous bases are 2’deoxyribose (no OH on the 2’), and we know synthesis of DNAis by extending the 3’end (need the 3’OH to extend) ○ The defective nitrogenous bases would thus be 2’, 3’dideoxy (no OH group on 2’ and 3’) ● What would normally happen when you add polymerase to the mix of plasmid species is that, with primers in place, polymerase would just go ahead and synthesize the single stranded plasmid pieces ● Now, adding in small amounts of dideoxynucleotides will terminate synthesis of some chains ● Eventually, you will have some chains that terminate at each position of each ddNTP ● Next, analyzing the products by gel electrophoresis allows us to see the full sequence of the plasmid by reading the sequence from the bottom to top (5’to 3’) ● Note, it is important you don’t put in too little of the ddNTPs as the chains wouldn’t stop synthesizing enough and the sequence could not be read ○ too much ddNTPs would result in the all the chains stopping too often and again the full sequence could not be read ● If you don’t understand this still, watch a video on it online, it’s a really clever process ● In the mid 1190s automated DNAsequencing became common place; each ddNTP is labeled with a different dye that fluoresces at a different wavelength ○ this is what they do today to sequence DNArather than running it on a gel Getting the Protein Product ● E.coli are the most commonly used organism for
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