Prelab+Questions

Note: I have changed the font color to white for all the answers - test yourself and highlight to see if you are correct. Also, I am missing some of my prelabs so the answers might not all be correct.

Exp 12: 2. How do Buffer A and Buffer B help you achieve this goal? (i.e. look at the components of the buffers and explain their presence). What does Ni-NTA stand for? Knowing the resin you are using, what is the composition of the “tag” on your protein? What is the purpose of imidazole? ** Imidazole is used to elute tagged proteins bound to Ni ions attached to the surface of beads in the chromatography column .** What does IPTG stand for? What is IPTG ? ** Isopropyl ß-D-1-thiogalactopyranoside, It is a chemical that can be used to mimic allolastose but cannot be metabolized by cells .**
 * Buffer A contains lysozyme which attacks peptidoglycans so I think it is safe to say that Buffer A destroys the e.colis’ cell walls. **** i **
 * Buffer B has multiple surfactants Tween and NP-40 these should make the cell membrane of the e.coli permeable . **
 * N﻿﻿ickel-nitrilotriacetic acid, polyhistadine . **

Exp 13: What is the purpose of each of the following named DNA samples? Fill in the table below identifying exactly the form(s) of DNA in each sample and what it will be used for (example: DP1000 genomic DNA: DNA isolated from bacteria that will be digested with EcoR1 to make genomic digest. It will be run on the gel). Indicate on the table which samples will be run on gel electrophoresis later? Be ligated into the plasmid. || Yes ||
 * **Sample name** || **Form and Use** || **Run on gel?** ||
 * DP1000 genomic DNA || DNA isolated from //Strep.pneunoniae//, the source of the DNA to
 * genomic digest || In this fraction the genomic DNA will be cut at ecoR1 sites. || Yes ||
 * plasmid DNA (pBlue) || The genomic DNA will be placed into these plasmids and transfected into e.coli for amplification. || Yes ||
 * diluted pBlue || This is the plasmid into which chunks of genomic DNA form //Strep.pneunoniae// will be transfected. || Yes ||
 * plasmid digest || In this fraction the plasmid will be cut at ecoR1 sites. || Yes ||
 * diluted plasmid digest || We want to have a ratio of plasmid and genomic DNA that keeps them from binding back to how they were before they were cleaved. ||  ||
 * ligation control || This allows us to see what unligated plasmid and DNA pieces look like || Yes ||
 * ligation || These are the stuff we want to transfect into e.coli` || yes ||

Where does the EcoR1 digest the pBluescript? How many pieces of pBluescript should result from the digestion? What should be the size(s) of the resulting piece(s)? **According to lablife.org the ecoR1 site starts at NA 701 and cuts before NA 702 ** [] Where does the EcoR1 digest the genomic DNA? How many pieces of genomic DNA should result from the digestion? What should be the size(s) of the resulting piece(s)? **EcoR1 will cleave at any site with the sequences ** **GAATTC **** or ****CTTAAG ****. It can also cleave non-specifically if ****low salt concentration ****s ****, high glycerol concentration ****s ****, ** **excessive amounts of enzyme ****are ****present, **** in ****high pH ****, ****<span style="font-family: Cambria,serif;"> and with certain organic solvents ****<span style="font-family: Cambria,serif;">. However they should be much more numerous than the plasmid and would be in a range of different sizes most should be larger than the plasmid. ** **.** You then do a ligation reaction with restriction-digested plasmid DNA and restriction-digested genomic DNA in the reaction mix. What product are you trying to make? Draw a map of the desired product, clearly indicating what is plasmid DNA and what is genomic DNA. Do you expect the product to be homogeneous (all the same) or heterogeneous? Why? **We are trying to make a plasmid with a piece of DNA from the genomic DNA inserted into it at the restriction site. These should be heterogeneous as we will have all kinds of various fragments from the genomic DNA.** **Drawing: The picture should basically show that the insert is much larger than the plasmid.** **.** How do you expect the EcoR1 restricted genomic DNA to run on the gel compared to the isolated genomic DNA? **<span style="font-family: Cambria,serif;"> The restricted DNA should be smaller. ** How do you expect the EcoR1 restricted pBluescript DNA to run on the gel compared to the supercoiled pBluescript DNA? ** It should be much smaller .** What is the purpose of the ethidium bromide in the gel? What safety precautions in handling and disposal are appropriate when using ethidium bromide? **<span style="font-family: Cambria,serif;"> It makes the DNA fluoresce under UV. Ethidium bromide is carcinogenic so we should wear gloves and dispose of it in the ethidium bromide waste. **

List the eight reagents, and the purpose of each, used on Day 1 for the isolation of genomic DNA. **1.1.** **DP1000 cell suspension** **1.2.** **10%SDS** **1.3.** **Proteinase K** **1.4.** **Phenol: chloroform: isoamyl alcohol** **1.5.** **Chloroform: isoamyl alcohol** **1.6.** **95% ethanol** **1.7.** **70% ethanol** **1.8.** **TE Buffer**

What safety precautions will you take today and why? **We will weat gloves and goggles, as phenol as we have seen in the safety video can cause burns.** How will you know if you have good quality genomic DNA? **If a stringy precipitate is visible after the final extraction.**

Exp 14: 1. What is meant by the term **transformation** as used in this experiment? ** The uptake and incorporation of foreign genetic material from the bacteria’s surroundings via the cell membrane. ** 2. **List** the characteristics of //E. coli// strain DH5 a that make it a good choice for the competent cells used in this experiment, and explain why each of these characteristics is important. **1.** **The endA1 mutation inactivates an intracellular endonuclease which degrades plasmid DNA.** **2.** **The hsdR17 mutation eliminates the restriction endonuclease from the EcoKI restriction-modification system, thus DNA lacking EcoKI methylation will not be degraded.** **3.** **(lacZ)M15 is the allele used for blue-white screening with many lacZ vectors.** **4.** **recA eliminates homologous recombination this reduces deletion formation and plasmid multimerization.** **5.** **glnV44 is the systematic name for SupE44, an amber suppressor** 3. What criteria will you use to choose colonies to grow up for plasmid DNA purification? Why do you need to streak these colonies on a second TAXI plate?

**The TAXI plate contains Thiamine, ampicillin, X-gal, and ITPG. Thiamine is required by DH5**** a ** ** for growth, ampicillin ensures that bacteria that do not have the plasmid do not grow., X-gal indicated the presense of B-galactose giving further information about the condition of the plasmid in the colonies, and IPTG induces beta-galactose. ** 4. Why is it necessary to have ampicillin in liquid culture medium used to grow the isolated clone for purification of the plasmid DNA? ** The plasmid contains the gene for ampicillin resistance so any bacteria that grow in the media will have transformed the plasmid. **

You will be using a QIAprep kit today. What is the goal of using this kit (ie. what are you starting with and what are you ending with)?

**<span style="color: black; font-family: Cambria,serif;">The QIAprep Kit is designed for isolation of up high-purity plasmid or cosmid DNA. We will use this to extract the plasmid DNA from the transformed e.coli. **

What is a nanodrop and why are we using it?

**A nanodrop is a spectrophotometer which is able to operate with very small volumes. This is very useful when doing experiments where large volumes of sample are not feasible or not needed.**

Exp 15: No prelabs for this lab

Exp 16: 1. What instructions must you follow before beginning today's lab?

**Do not eat or drink for 30 minutes before today’s lab.**

2. What is the purpose of each component in the Master Mix used in this PCR experiment?

· 10x PCR buffer – **buffer to stabilize reaction, kind of self explanatory** · 25mM MgCl2 – **Mg2+ is a cofactor of Taq polymerase** · 10mM dNTP mix - **Deoxynucleotide triphosphates, these are the building-blocks from which the DNA polymerase synthesizes a new DNA strand.** · Primer 1/ Primer 2 – **The sense/anti-sense pair, these bind to the corresponding pieces of single stranded DNA, providing a starting place for the DNA polymerase.**

3. Why do we use a master mix rather than pipetting each component separately?

**We use the master mix to reduce the variation between tubes and reduce the chances of the samples being contaminated.**

1. How must we treat the PCR product after storing it frozen before running the gels?

**We must remove 15ul add 5ul of loading buffer and heat the sample 65C for ten minutes.**