Victoria Buchan- Phage Lab Fall 2015

Transcription

1 Victoria Buchan- Phage Lab Fall 2015 SKILL Pipetting Aseptic Technique DATA (See Lab Manual for methods.) Always flame the glassware you are using and make sure to work close to the flame to ensure no particles fall into and contaminate your sample. Date of Collection: 24 August 2015 Temperature at Time of Collection: 85 degrees Fahrenheit Soil Sample Collection GPS & Location: N37 32' , W77 25' Duval Street Connector, Richmond,VA NOTE: Sample has a very grainy appearance (sort of like a sand/dirt mixture) and includes some mulch and grass Circle One: Identification of Plaque Direct Plating Enrichment Plating Phage Name: Purification of Plaque Number of serial dilutions performed: 7 Describe Final Plaque Morphology Production of Web Plate Isolation of High-titer Lysate Clear/ Turbid/ Halo/ Comet/ Size/ Multiple Empirical/ Intuitive Titer: DNA Purification, How many columns did you use? DNA Quantification, Concentration/Volume? Restriction digest, Which enzymes cut? EM Visualization Head Diameter: Tail Length: 25 August 2015 Today we are setting up an enrichment culture using 45mL of T-soy Broth and 1mL of the Bacillus Thuringiensis (subspecies: Kurstaki). We began with 46mL total. The sample will be incubated for 24 hours at 30 degrees, shaking at 180rpm. We also practiced serial dilutions with water by using blue dye to show the degree of dilution. (10x dilution each time) 100mL of undiluted blue dye is in the 10^0 tube. (10^0 being the most concentrated) 90mL of water is in each of the other tubes. (10^-4 being the least concentrated) Pipette 10mL from the 10^0 tube into the 10^-1 tube, and then 10mL from the 10^-1 tube into the 10^-2 tube, and so on... Next class: We will filter our culture and use the Enrichment plating technique. AJ August 2015 Today we are using the Phage Filtrate and Enrichment plating techniques to see if we have a phage in our soil sample.

2 After spinning in the Centrifuge (21 degrees Celsius at 2,000rpm for 5 minutes), our culture is ready to be filtered. We set up the bacteria in the agar plate by giving the bacteria liquefied agar as food and letting the plate set up. While the plate sets up, we filtered our culture using a syringe and screw on filter (0.22 um) to putting just a few drops into a tube (this is our 100). We then used the serial dilution technique with phage buffer instead of water (see 8/25) to After serial dilution, we used a pipette to put 5uL from each tube onto their own section of the agar plate. We are letting the agar plate set to see if there are any phage growing. Plate will be incubated to 30 degrees, over night. (plate is labeled Spot Test 1) Next class: We are doing our first round of purification if there is phage growing in our agar plates. Make sure to take pictures! If there is no phage, we will use the direct plating method with a new sample and try again! AJ September 2015 Purification Round 1

3 Results: Success! We have phage!! Plaques are shown in the 10^0 and 10^-1 spaces. They are very cloudy but is still visible. (Plaques from left to right in image 1 are 0.7cm, 0.6cm, and 1cm) Although there was success, we are going to re-filter our sample and try to get a more clear plaque. (because class culture was contaminated, see filtration technique above; 08/27/15) We are trying again but this time each dilution will have it's own plate ~ 10^0 on one plate, 10^-1 on one plate, 10^-2 on one plate, 10^-3 on one plate, 10^-4 on one plate After filtration and dilution, we put 10uL from each dilution into their own tubes with the BTK Bacteria We then let the tubes set for about 5 minutes to allow infection before adding agar to the tubes. Immediately after adding the agar, we put each tube onto their own plate (as stated above). Plates are going to be put in the in the incubator over night at 30 degrees. Next Class: We are going to count the amount of phage present and do the next round of purification. AJ September 2015 Purification Round 2 Results: More success with our round of purification. There are many more phage visible in the 10^0 and 10^-1 plates, however the cultures seem to be contaminated. There seems to be a single uncontaminated spot on the 10^-3 plate. (In the images, plates stay in the same order throughout to ensure that consistent data is recorded.) Plates 10^0 and 10^-1 have a very glossed, cloudy appearance. Plate 10^-3 has a single small, clear spot with a tiny black dot in the middle of it.

4 Today we are only recording data and placing the plates back in the refrigerator to continue to grow. Next Class: We are going to do another round of purification on our plates. Kaivalya Dandamudi 9/3/15 15 September 2015 Purification round 3 Today I am going to try serial dilution again and re-plate because the last culture seemed to be contaminated. I will use the same materials and methods as before. (See previous notes.) The plates will be put in the incubator over night at 30 degrees. Filtration Serial Dilution Infection Plating Next Class: We are going to continue purification by either the stick streak method or plaque pick, serial dilution, infection, and plating. Tom Mathew 09/15/15 17 September 2015 Purification Round 4 Results: There are some definite plaques on almost all of the plates. Plaques are about 0.10cm in diameter.

5 Today I am going to prepare two plates by stick streak method. > Stick Streak ---- Then pour top agar mixed with BTK bacteria onto the plate and let the plate set up. (Pour from lowest to highest concentration of phage.) Touch the center of a well isolated plaque using a sterile micropipette tip. (I picked off of my 10^-1 plate from last class.) Transfer the micropipette tip to Phage Buffer (PB) and mix. Flame the loop, dip in the PB-plaque mixture, and streak the first portion of the agar plate. Flame loop -> 2nd streak -> flame loop -> 3rd streak (Make sure the streaks do NOT overlap!) We are going to put the plates in the incubator over night at 30 degrees. Next Class: We are going to continue with the dilution process. Kaivalya Dandamudi 09/17/15 29 September 2015 Results: It looks as if the stick streak method was unsuccessful. There do not seem to be any phage on the plates. Methods used today: Today I am going to pick another plaque off of my 10^-1 plate and mix with the BTK bacteria. --> I'm going to wait 45 minutes to allow infection before plating the new culture.

6 I am also going to try another stick streak using a plaque from the 10^-1 plate Note: I am using two different plaques from the 10^-1 plate made on 09/15/15. Next Class: We are going to continue with the next round. AJ October 2015 Purification Round 5 Results: Again the stick streak was unsuccessful. The infection however seemed to be quite successful. (See pictures below.) There seem to be many isolated plaques and most are about 0.2cm-0.3cm in diameter. All plaques are cloudy but visible.

7 Today I am going to complete the Phage-Titer Assay by: tip dip --> into phage buffer --> serial dilution --> infection (20 minutes this time) --> make 5 plates I will be picking my plaque off of the successful plate from last class (09/29) (Pictured above). After serial dilution, I will save my 10^0 tube in the fridge just in case today was unsuccessful. Plates are going to be put into the incubator over night at 30 degrees. Note: See previous notes and lab manual on procedures. Next Class: We are going to continue purification. Kaivalya Dandamudi, 10/1/15 06 October 2015 Purification Round 6 Results: Success on my 10^-2 plate! Plaques are visible! Plaques have mixed morphology and sizes range from >0.1cm to 0.3cm in diameter (1-3 mm).

8 *Some plaques are turbid (hazy), some clear, and some have halos. This indicates mixed morphology.* Plates 10^0 and 10^-1 have been lysed with very little lawn left around it. Plates 10^-3 and 10^-4 seem to have no results. Today I am going to make three stick streak plates, one for each type of morphology. (See previous notes and lab manual for methods.) I am going to use 100uL of phage buffer this time. Plates will be labeled with the type of morphology the plaque was before stick streak. Plates will be put in the incubator over night at 30 degrees. Next Class: I am looking for consistent morphology in my phage. If the plaques still have mixed morphology then that is the behavior of my phage. I am going to continue with whichever plaque comes out the best. Tom Mathew 10/06/ October 2015 Purification Round 7 Results: It seems that the stick streaks was unsuccessful. No phage appear on the plates. (Plaques) However, the plaque morphology on my 10^-2 plate ( 10/01) seems to be more similar now that it has been in the fridge for a longer period of time. I see more cloudy than halo or clear. Today I am going to pick just one type of plaque (halo?) from the plate stated above and do a serial dilution to 10^-2, infection for 20 minutes, and plating (see previous notes and lab manual). Next class: I am going to continue with purification- last round? AJ October 2015 Results: After picking a halo plaque and completing the process stated above, it seems the plaques have mixed morphology. Plaques seem to be about an average of 0.2cm in diameter.

9 Today I am going to move ahead to the Empirical Testing calculation. (Shown here: Empirical Testing.ppt) (90^2)/(2^2)=2,025 (32 plaques counted)/10ul x 1000uL/mL x 10^2 = 3.2 x 10^5 pfu/ml 2,025/(3.2 x 10^5) = mL = 6.3uL = 63ul of 10^-1 My web plate is 10^0 (only needed a 10^-2 dilution). Next class: I am going to complete empirical testing. TA: Parantap Patel 20 October 2015 Today I am going to use my 10^-1 plate from 10/13 and tip dip into 100uL or phage buffer (this will be my 10^0). I am then going to take 50uL of that and put into 450uL of phage buffer (this will be my 10^-1). (5X) --> I am going to take 63uL of my 10^-1 and put into BTK for about 20 minutes to allow infection. Then I will plate and put into the incubator over night at 30 degrees. Next class: I am supposed begin DNA preparation. Tom Mathew 10/20/ October 2015 Results: All five plates came out very successful in terms of having plaques, however they were not webbed plates. Plaques are visible on every single plate. plaques are about 1mm in diameter.

10 Today I am going to try the same procedure as last class (10/20) to get a new five plate infection, but I am going to do a normal tip dip into 100ul of phage buffer and transfer 33uL into 297uL for my 10^-1, allow 63uL to infect 5 separate cultures for 20 minutes, and make five plates. Next Class: I am going to come in and collect my high titer lysate, stain for electron microscopy, and set up a phage precipitation for DNA purification. AJ October 2015 Results: It seems that my plates look the same as the results from last class. There are visible plaques (about 1mm each in diameter) on every plate but none of the plates are webbed. Today I am going to try to get a new five plate infection by doing a normal tip dip (from one of my plates from 10/22) into 100uL of phage buffer and transfer 20uL into each BTK culture (infect for 20 minutes), and make five plates. Next Class: I am going to collect my high titer lysate, stain for electron microscopy, and set up a phage precipitation for DNA purification. Signed by Malika Gill Add plate pics 29 October 2015 Absent: Phage lysate was collected for me by the TA's. 03 November 2015 Today I am going to do a serial dilution to the 10^-7 with the high titer lysate from 10/29 and make eight plates with BTK bacteria. This is to calculate the titer, or concentration of phage, in my high titer lysate. I am also going to stain for electron microscopy and create three archive tubes. ( See Lab Manual for procedures) I set up a phage precipitation with ~10 ml of high titer lysate + 40 ul nuclease (degrades bacterial DNA and RNA). Allison will add phage precipitate solution after 1.5 hours and centrifuge the sample before class on Thursday to form a phage pellet. Next class: I am going to do DNA purification. AJ November 2015 Results: There is a small pellet in the tube from last class. Also, my plates seem to have no phage on them. I may have to try a second time to get phage that I am able to count.

11 Today I am going to do DNA purification by: Add 500 ul of sterile ddh2o to the pellet and gently re-suspend the pellet by pipetting up and down. Apply 2000 ul of water-resin phage-genomic-dna solution to each column using a pipette. Add 0.5 ml of 80% isopropanol to the column. Spin columns in a microcentrifuge at 13,000 rpm for one minute. Apply 50 L of pre-warmed, 80 degrees Celsius H2O to the resin in the column. Let the H2O sit on the column 30 to 60 seconds to release the DNA. Spin columns in a microcentrifuge at 13,000 rpm for one minute to collect DNA. (Put samples into a single tube and store at 4 degrees Celsius.) NOTE: DNA sample was put into spectrophotometer and concentration was finalized at ng/ul Next Class: I am going to set up for Restriction Digest. Tom Mathew 11/05/15 19 November 2015 DNA Concentration = ng/ul 737.3/1 = 500/x x= 1.47uL= 1.5uL Solution: Tube 1 Tube 2 Tube 3 Tube 4 Tube 5 10X reaction buffer 2 l Buffer 3 2 l Buffer 3 2 l Buffer 4 2 l Buffer EcoRI 2 l Buffer 2 Phage Genomic DNA 1.5 l 1.5 l 1.5 l 1.5 l 1.5 l 10X BSA 2 l 2 l 2 l 2 l 2 l BamHI 3 2 l ClaI 0.5 l EcoRI 2 l HindIII 2 l ddh O l 12.5 l 14 l 12.5 l 12.5 l Total: 20 l 20 l 20 l 20 l 20 l Today I am going to do restriction digest. I am going to have five different tubes with 2uL of reaction buffer in each tube. I am going to add 1.5uL of my phage genomic DNA and

12 2uL of BSA. Each tube will have a total of 20uL. Tube 1 will have 14.5 ul of ddh2o4 ; Tube 2 will have 2uL of BamHI3 and 12.5uL of ddh2o4 ; Tube 3 will have 0.5uL of ClaI and 14uL of ddh2o4 ; Tube 4 will have 2uL of EcoRI and 12.5uL of ddh2o4 ; Tube 5 will have 2uL of HindIII and 12.5uL of ddh2o4. I will pipette water, buffer, BSA, enzyme, and DNA into the tubes in that order! I am also going to stain a gel to show which restriction enzymes cut the DNA.