Lab 6 - Overview

Lab 6 Overview and Steps 1-3 Learning Objectives **This is what our first 5 labs have been building towards! Using aseptic technique as needed, we will: 1. 2. 3. 4. 5. 6. 7. 8. Cut two plasmids with restriction enzymes Join the fragments into random combinations using ligase (*Ligation is new) Transform E. coli with your various plasmids Select a colony that is resistant to both antibiotics Isolate the plasmids from that colony (*Also a new technique) Quantify your DNA (*New) Digest the plasmid DNA one final time View and analyze results with gel electrophoresis Step 1: Restriction Digest What are our plasmids? What restriction enzymes will we use? What sorts of combinations could be made? Will every combination successfully replicate? Will every combination result in antibiotic resistance? Like previous restriction digest, we’ll be adding plasmid, buffer, and enzyme together: How many total micrograms of DNA will we start with (per tube)? How many restriction enzymes are we adding? Why? Why keep the plasmids in separate tubes? Tube pAMP pKAN 0.2 ug/ul 0.2 ug/ul 2X Buffer BamHI/ HindIII pAMP (digested) 5.5 ul* - 7.5 ul 2 ul pKAN (digested) - 5.5 ul* 7.5 ul 2 ul Step 2: Ligation Where do the digested plasmids come from? Why heat both tubes to 65 degrees Celsius? Why does the ligation buffer contain ATP? What sort of bonds does ligase make? Do you think you’ll need to spin this reaction down? With what tool? Why a room temperature incubation? Methods: Ligate pAMP and pKAN 1. Incubate the tubes labeled Digested pAMP and pKAN (from Part A) in a 65°C water bath for 10 minutes. This step is critical. Heat denatures proteins, thus inactivating the restriction enzymes. 2. Label a clean 1.5 ml tube LIG (for ligation). Include your initials (top and side). 3. Use the matrix below as a checklist while adding reagents to the LIG tube. Use a fresh tip for each reagent. Refer to the detailed directions that follow. 4. Materials: Reagents Supplies and Equipment Digested pAMP (from Part A) Digested pKAN (from Part A) 2X Ligation Buffer/ATP Distilled Water T4 DNA ligase water bath set at 65°C 1.5 ml tube Micropipettor and tips 5. 6. 7. 8. 9. 10. 11. Tube Disgested pAMP Digested pKAN 2X Ligation Buffer/ATP Water Ligase LIG 3 ul 3 ul 10 ul 3 ul 1 ul Collect reagents (except ligase) and place them in a test tube rack on the lab bench. Add 3 ul of Digested pAMP to the LIG tube. Use a fresh tip to add 3 ul of Digested pKAN to the LIG tube. Use a fresh tip to add 10 ul of 2x Ligation Buffer /ATP to the LIG tube. Use a fresh tip to add 3 ul of distilled water to the LIG tube. Let your teacher know you are ready for the T4 ligase. She will bring you your 1 ul of ligase. Close the tube cap. Pool the reagents by pulse spinning in a microcentrifuge. Place your LIG tube in the designated rack on the benchtop. Incubate the reaction overnight at room temperature. Prep for step 3: Creating Competent Cells MM294 E. coli grow overnight then are added to a flask of sterile LB for 1.5-2 hours. Cells should now be in mid-log phase. At this point, you can follow the diagram. A series of centrifugation steps, additions of ice-cold CaCl2, finger flicking to vortex, and ice incubations result in cells that should be ready for transformation. Step 3: Transformation What are some similarities and differences between these transformation steps and our steps during lab 5? Step 3: Transformation Ligated DNA is more likely to be in a relaxed circular or linear form (rather than supercoiled like our previous plasmids) Transformation will be less efficient. pAMP and pKAN will be used as controls (by your teachers) The recovery step is essential for cell survival and growth when plating on kanamycin. To have time for all these steps, you need to be prepared and aware. Steps 4-8 Step 4 - Colony Screening ● Remember the end goal: ○ Determine which fragments were ligated together to make a recombinant plasmid resistant to both ampicillin and kanamycin ● First step - isolate a colony from the LB + amp + kan plate ○ Why a single colony? ● Next step - grow up genetic clones in 2 mL of LB + amp + kan broth ○ Why both antibiotics? Step 4 - Colony Screening ● How will we grow the culture? ○ At 37 degrees C ○ Overnight ○ In a shaking incubator (constant motion allows aeration for optimal growth) Step 5 - Miniprep (overview of steps) Protein digestion, genomic DNA removal - spin take supernatant plasmid Binds Plasmid DNA Notice the change in tubes. The plasmid DNA will be caught in the filter of an insert that can be moved to new tubes. Here is your DNA (Released from the filter into a clean tube.) Step 5 - Miniprep (What can you do with mostly clean, pure DNA?) Sequencing Step 5 Mini-prep ● ● ● ● All mini-preps are complex processes with multiple steps Centrifugation steps vary by gravity and time Samples move from tube to tube Sample of interest might be in your supernatant, your pellet, your filter, etc… BE CAREFUL (pathlength) Step 6 - Quantifying Plasmid DNA Before using DNA samples in analytical techniques, the quality and usability must be determined through DNA quality indicators, which include DNA purity and integrity. The ratio of absorbance at 260 and 280 nm is used to assess DNA purity. A ratio of ∼1.8 is generally accepted as “pure” for DNA. If the ratio is appreciably lower (≤1.6), it may indicate the presence of proteins, phenol, or other contaminants that absorb strongly at or near 280 nm. To the contrary, since absorbance readings cannot discriminate between DNA and RNA, the presence of RNA can lead to the ratio increasing and this possibility must be considered to avoid DNA over quantification 2016 - DNA Source Selection for Downstream Applications Based on DNA Quality Indicators Analysis Step 6 - Quantifying Plasmid DNA ● ● ● ● ● ● We have a UV-vis microspectrophotometer that allows us to quantify DNA in samples 2-5 uL 260nm determines the amount of nucleic acid in the sample 280nm indicates the amount of protein 260/280 ratio will indicate purity of the sample DNA should have a 1.8 reading RNA should have a 2.0 reading Step 6 continued Our step 5 elution could be used for many downstream applications. This special Take3 plate allows us to conserve our DNA by only using 2 uL samples when we quantify. Step 7 - Restriction Digest ● What have we just extracted from the E. coli colony screening? ○ Recombinant (amp + kan) plasmid ● Why would we digest it? ○ To see what fragments make up the larger plasmid ● How will we digest it? ○ The same enzymes we used previously ○ HindIII and BamHI Step 8 - Gel Electrophoresis ● You will run a final gel with the following: 1. Ladder 2. pAMP (digested) 3. pKAN (digested) 4. Ligation reaction 5. Plasmid from mini-prep (digested) 6. Plasmid from mini-prep (undigested) *Control Microprep Reminders Microprep Step Pellet Supernanant Cell Harvest Bacterial Cells / KEEP LB Broth / TOSS Lysis Genomic DNA, Proteins / TOSS Plasmid DNA / KEEP Wash Filter has Plasmid DNA / KEEP Tube has waste from rinse / TOSS Elute Filter Column releases Plasmid/ TOSS Tube has Plasmid and Buffer / KEEP Ideal gel: pAmp pKan Ideal gel: pAmp pKan pLIG extracted plasmid extracted plasmid (digested) (undigested control)