Difference between revisions of "Team:IISc-Bangalore/Notebook"

Line 49: Line 49:
  
 
<h2>29/3/17</h2>
 
<h2>29/3/17</h2>
 +
<p>Plasmid (Yeast GFP) brought from Prof. Rajyaguru’s lab</p>
 +
<p>pRS316 Amp/Ura. High conc, use 1 μL from Nupur’s protocol</p>
 +
<p>Will show GFP expression in yeast only, not E.coli</p>
 +
 +
<h2>31/3/17</h2>
 +
<p>Buffers for electrophoresis were made and autoclaved</p>
 +
<p>Electrophoresis showed presence of plasmid! :D </p>
  
 +
<h2>02/4/17</h2>
 +
<p>YPD media prepared and inoculation done.
 +
 +
<h2>03/4/17</h2>
 +
<p>Showckwave transformation canceled by Akshay. Images of yeast cells were taekn under the microscope for heamocytometry</p>
 +
<p>At night comp cells prep was done. Gel was run for the transformants (terrible results, needs redoing)</p>
 +
 +
<h2>30/4/17</h2>
 +
<p>Lab meeting done at 20:00. Plans were laid down for the whole project. As a whole, need to
 +
<ul>
 +
<li>Overexpress hexose transporters
 +
<li>Overexpress invertase
 +
<li>ACE2 gene knockout to induce clumping
 +
<li>Create GFP-GVNP and GVNP-TEV biobricks
 +
<li>Hardware part
 +
<li>Fiji plugin for haemocytometer yeast count
 +
<ul>
 +
</p>
 +
<p>Fresh LB, YPAD, YPAD (2x) was prepared</p>
 +
 +
 +
<h2>01/5/17</h2>
 +
<p>Heat shock transformation carried out in Dipa lab.
 +
 +
<h2>02/5/17</h2>
 +
<p>(DC lab) Observation:- The colonies look good. Thus procedure shall be taken forward. Fresh media prepared, plates autoclaved.</p>
 +
 +
<h2>04/5/17</h2>
 +
<p>Raj and Sharath arried out miniprep in DC lab.</p>
 +
<p>Nanodrop readings too high. Ran, gel; too thick, not too clear. Repeated gel with lower concentration of DNA, presence of genomic DNA contamination and RNA contamination (probably non functional RNAse), need to repeat experiment</p>
 +
 +
<h2>05/5/17</h2>
 +
<p>Raj and Sharath repeat miniprep, ran gel. Do get plasmid bands but with heavy DNA adn RNA contamination (except for the third lane, for reasons unknown)</p>
 +
<p>SOB plates were prepared, YPAD plates were prepared, and some required reagent solutions were prepared</p>
 +
 +
<h2>07/5/17</h2>
 +
<p>YEast cultures discarded, cant store them  properly without drastically affecting the effciency. Fresh incoculum prepared</p>
 +
 +
<h2>08/5/17</h2>
 +
<p>Bulk extraction commences from a 50 ml culture. Ran the gel and SUCCESS! :D . Realized some errors in our protocol which helped us prevent genomic DNA contamination. Nanodrop readings were positive and resuspension was done in TE and not MilliQ. </p>
 +
 +
<h2>09/5/17</h2>
 +
<p>Met Nupur and asked for selection media, and the compelte plasmid map we were given. Alos shockwave transformation is a go, the first trial shoould be over tomorrow, 10th</p>
 +
 +
<h2>10/5/17</h2>
 +
<p>Cleaned up the lab: hoods, drawers, trash. Rearranged equipment more sanely. Checked stocks of Eppendorfs, tips, etc.</p>
 +
 +
<h2>11/5/17</h2>
 +
<p>LiAc transformation and shockwave transformation were carried out. Technical difficulties lead to LiAc transformation taking a LOT of time to carry out. </p>
 +
<p>Media was prepared</p>
 +
<p>After meeting with Dr Srinath, realized "Why work with yeast? <i>E.Coli<i> is easier to work with, protocols are easier and less time consuming"</p>
 +
 +
<h2>12/5/17</h2>
 +
<p>Made reagent stocks.</p>
 +
 +
<h2>15/5/17</h2>
 +
<p>Media preparation. Different percentages of agarose gel prepared to check for fragility and suitability for low melting agar for cell density measurements. The burette idea was discarded, because vortexes for amd mixing occurs.</p>
 +
 +
<h2>16/5/17</h2>
 +
<p>Inoculated DH5A (no plasmid) in LB media. Realized there are some contamination problems. Glycerol stocks were prepared and some transformants were plated</p>
 +
 +
<h2>18/5/17</h2>
 +
<p>More inoculations done, growth curve of <i>E.coli<i> and yeast was carried out.</p>
 +
<p>At 4:35 PM Srinath sir entered the lab and questioned everyone about the project. He pointed out various errors people were committing and asked for a lab meeting scheduled on 19th.</p>
 +
 +
<h2>30/5/17</h2>
 +
<p>The lab notebook had been neglected for the past few days, sadly</p>
 +
<p>Yesterday, five different transformants of pSB1A3 (ampicillin resistant) were plated. Today, we will miniprep the plasmid and ideally run a gel to confirm. To do this, we have prepared alkaline lysis solutions I and III beforehand.</p>
 +
<p>Made workling stocks of the required reagents</p>
 +
<p>Ran a miniprep. Ran a gel, absolutely no bands were present. There was a minor screwup while loading. Will run gel again tommorrow.</p>
 +
 +
<h2>05/6/17</h2>
 +
<p>Once again, a major ga in the lab notebook.
 +
<p>Possible reason for failure in gel- maybe used 50x TAE rather than 1x. Maybe EtBr diffused too fast. The second gel ran on 31st failed because probably 20x SB was used. . Even the ladder was not seen so the problem lied with the gel. The third gel maybe ran for too long and thus no bands were seen. </p>
 +
<p>On the plus side, the assembly plan is going well. Primers are being designed, to be finalized with Arunavo tomorrow and then with Srinath sir afterwards</p>
 +
<p>Four primary inoculums made today. If they are dense enough then we'll carry out miniprep tomorrow, then run a gel and discuss primers.</p>
 +
 +
<h2>08/6/17</h2>
 +
  
  

Revision as of 11:40, 1 November 2017

Preparation

16/3/17

Autoclaved items and prepared media

17/3/17

Obtained S. cerevisiae BY4741 from Dr. Rajyaguru’s lab and incubated it at 30 degree celcius

Inoculated the above strain in 2 mL YPD [4.5 mL YPD media + 0.5 mL 20% glucose] using a sterile toothpick

18/3/17

Went to Prof. Dighe’s lab to get some chemicals, sans people

Yeast in the inoculated tube are growing fine but seem to have aggregated at the bottom.

19/3/17

Started 8 growth curves

Cuvette placement (orientation) was not right for the first few readings; path length was not 1 cm.

21/3/17

Autoclaved plates, falcons, media (YPDA and YPGA)

Got the plasmid pRS316 and HeLa cells from a lab

Decided to try shockwave transformation of yeast. We will be amplifying the plasmid pRS316 in E. coli, mini prepping it and taking it to Aerospace department.

22/3/17

Got DH5a competent cells from Prof. Chakravortty’s Lab. We have transformed the pRS316 plasmid in E. coli. We also made SOB media. Our selection marker for now is Ampicillin.

23/3/17

Plates (one streak and one spread) yeast culture and kept the remaining in cold room. We also observed plates of E.coli DH5a today

24/3/17

Transformants from the Amp+ plate were inoculated in SOB (3 test tubes)

WT from the amp- plate was inoculated in SOB (2 test tubes)

Observations: No GFP fluorescence was observed, possibly because plasmid is low copy (?). Note from Sai: In hindsight, we should have realized that the GFP was under a yeast promoter (hehe)

27/3/17

Step 8 of protocol failed: No pellet was obtained. Need to redo entire set of experiments :(

Miniprep was then carried out (protocol follo9wed from Sambrook

29/3/17

Plasmid (Yeast GFP) brought from Prof. Rajyaguru’s lab

pRS316 Amp/Ura. High conc, use 1 μL from Nupur’s protocol

Will show GFP expression in yeast only, not E.coli

31/3/17

Buffers for electrophoresis were made and autoclaved

Electrophoresis showed presence of plasmid! :D

02/4/17

YPD media prepared and inoculation done.

03/4/17

Showckwave transformation canceled by Akshay. Images of yeast cells were taekn under the microscope for heamocytometry

At night comp cells prep was done. Gel was run for the transformants (terrible results, needs redoing)

30/4/17

Lab meeting done at 20:00. Plans were laid down for the whole project. As a whole, need to

  • Overexpress hexose transporters
  • Overexpress invertase
  • ACE2 gene knockout to induce clumping
  • Create GFP-GVNP and GVNP-TEV biobricks
  • Hardware part
  • Fiji plugin for haemocytometer yeast count

      Fresh LB, YPAD, YPAD (2x) was prepared

      01/5/17

      Heat shock transformation carried out in Dipa lab.

      02/5/17

      (DC lab) Observation:- The colonies look good. Thus procedure shall be taken forward. Fresh media prepared, plates autoclaved.

      04/5/17

      Raj and Sharath arried out miniprep in DC lab.

      Nanodrop readings too high. Ran, gel; too thick, not too clear. Repeated gel with lower concentration of DNA, presence of genomic DNA contamination and RNA contamination (probably non functional RNAse), need to repeat experiment

      05/5/17

      Raj and Sharath repeat miniprep, ran gel. Do get plasmid bands but with heavy DNA adn RNA contamination (except for the third lane, for reasons unknown)

      SOB plates were prepared, YPAD plates were prepared, and some required reagent solutions were prepared

      07/5/17

      YEast cultures discarded, cant store them properly without drastically affecting the effciency. Fresh incoculum prepared

      08/5/17

      Bulk extraction commences from a 50 ml culture. Ran the gel and SUCCESS! :D . Realized some errors in our protocol which helped us prevent genomic DNA contamination. Nanodrop readings were positive and resuspension was done in TE and not MilliQ.

      09/5/17

      Met Nupur and asked for selection media, and the compelte plasmid map we were given. Alos shockwave transformation is a go, the first trial shoould be over tomorrow, 10th

      10/5/17

      Cleaned up the lab: hoods, drawers, trash. Rearranged equipment more sanely. Checked stocks of Eppendorfs, tips, etc.

      11/5/17

      LiAc transformation and shockwave transformation were carried out. Technical difficulties lead to LiAc transformation taking a LOT of time to carry out.

      Media was prepared

      After meeting with Dr Srinath, realized "Why work with yeast? E.Coli is easier to work with, protocols are easier and less time consuming"

      12/5/17

      Made reagent stocks.

      15/5/17

      Media preparation. Different percentages of agarose gel prepared to check for fragility and suitability for low melting agar for cell density measurements. The burette idea was discarded, because vortexes for amd mixing occurs.

      16/5/17

      Inoculated DH5A (no plasmid) in LB media. Realized there are some contamination problems. Glycerol stocks were prepared and some transformants were plated

      18/5/17

      More inoculations done, growth curve of E.coli and yeast was carried out.

      At 4:35 PM Srinath sir entered the lab and questioned everyone about the project. He pointed out various errors people were committing and asked for a lab meeting scheduled on 19th.

      30/5/17

      The lab notebook had been neglected for the past few days, sadly

      Yesterday, five different transformants of pSB1A3 (ampicillin resistant) were plated. Today, we will miniprep the plasmid and ideally run a gel to confirm. To do this, we have prepared alkaline lysis solutions I and III beforehand.

      Made workling stocks of the required reagents

      Ran a miniprep. Ran a gel, absolutely no bands were present. There was a minor screwup while loading. Will run gel again tommorrow.

      05/6/17

      Once again, a major ga in the lab notebook.

      Possible reason for failure in gel- maybe used 50x TAE rather than 1x. Maybe EtBr diffused too fast. The second gel ran on 31st failed because probably 20x SB was used. . Even the ladder was not seen so the problem lied with the gel. The third gel maybe ran for too long and thus no bands were seen.

      On the plus side, the assembly plan is going well. Primers are being designed, to be finalized with Arunavo tomorrow and then with Srinath sir afterwards

      Four primary inoculums made today. If they are dense enough then we'll carry out miniprep tomorrow, then run a gel and discuss primers.

      08/6/17

      19/9/2017

      Prepared

      • 500 ml LB
      • 400+200 ml LB Agar
      • 32 petri plates
      • 2x LB plates

      Inoculated
      • DH5a in ml SOB (TSS comp cell prep)

      20/9/17

      1:30 AM

      • Set everything into autoclave

      3:00 AM
      • Took everything out of the autoclave
      • Plates dried for 1 hr, agar stored in oven

      4:00 AM
      • Plates dried in hood for 1 hr

      5:00 AM to 7:00 AM
      • Plates poured
      • Cmp stock conc = 35 mg/ml, working conc = 35 ug/ml

      21:30
      • TSS Protocol for comp cell prep

      22:10
      • Set secondary culture

      21/9/17

      Optimizing PCR (set 1)

      • Made reaction mixture of 200 ul per template , for splitting into 8 * 25 ul reactions
      • PCR reaction mixture
        • FP – 4ul
        • RP – 4ul
        • Stock template – 8 ul
        • 2x MM – 100 ul
        • ddH2O – 84 ul
      • Template stock – 100 ng/ul
      • Ran PCR 9:30 PM
      • Ran products on 1% agarose TAE gel (22/9, 12:20 AM)

      22/9/17

      Optimizing PCR (set 2)

      • Made reaction mixture of 200 ul per template , for splitting into 8 * 25 ul reactions
      • PCR reaction mixture
        • FP – 4ul
        • RP – 4ul
        • Stock template – 8 ul
        • 2x MM – 100 ul
        • ddH2O – 84 ul
      • Template stock – 100 ng/ul
      • Ran PCR (23/9, 12 AM)
      • Ran products on 1% agarose SB gel (23/9, 11 PM)

      23/9/17

      Pooling PCR (set 2)

      • Sai - PCR 4, Sharath PCR 3
      • Made reaction mixture of 1000 ul per template , for splitting into 20 * 50 ul reactions
      • PCR reaction mixture
        • FP – 20ul
        • RP – 20ul
        • Stock template – 40 ul
        • 2x MM – 500 ul
        • ddH2O – 420 ul
      • Template stock – 100 ng/ul
      • Ran PCR (24/9 , 2:00 AM) and pooled products
      • Ran products on 1% agarose SB gel (24/9, 4:00 AM)

      24/9/17

      Pooling PCR 1

      • Made reaction mixture of 1000 ul per template , for splitting into 20 * 50 ul reactions
      • PCR reaction mixture
        • FP – 20ul
        • RP – 20ul
        • Stock template – 40 ul
        • 2x MM – 500 ul
        • ddH2O – 420 ul
      • Template stock – 100 ng/ul
      • Ran PCR (24/9 , 20:00) and pooled products
      • Ran products on 1% agarose SB gel (25/9, 00:30)

      26/9/17

      Optimizing PCR 2

      • Made reaction mixture of 160 ul per template , for splitting into 8 * 20 ul reactions
      • PCR reaction mixture (Q5)
        • FP – 3.2ul
        • RP – 3.2ul
        • Stock template – 6.4 ul
        • 2x MM – 80 ul
        • ddH2O – 67.2 ul
      • PCR reaction mixture (Phu)
        • FP – 8ul
        • RP – 8ul
        • dNTP - 3.2ul
        • Stock template – 0.4 ul
        • Buffer - 32 ul
        • Phu pol - 1.6 ul
        • ddH2O – 106.8 ul
      • Template stock – 100 ng/ul
      • Ran PCR (26/9, 11:50 PM)
      • Ran products on 1% agarose SB gel (27/9, 3 AM)


      Interlab Day 1:-

      • Prepare 8 plates of Cmp LB agar plates
      • Plate the following
        • Positive control –B
        • Negative control – D
        • Device 1 – F
        • Device 2 – H
        • Device 3 – J
        • Device 4 – L
        • Device 5 – N
        • Device 6 - P

      27/9/17

      Pooling PCR 2

      • Made reaction mixture of 1000 ul per template , for splitting into 20 * 50 ul reactions
      • PCR reaction mixture (Phu)
        • FP – 50ul
        • RP – 50ul
        • dNTP - 20ul
        • Stock template – 2.5 ul
        • Buffer - 200 ul
        • Phu pol - 10 ul
        • ddH2O – 667.5 ul
      • Template stock – 100 ng/ul
      • Ran PCR ( 27/9, 9:30 AM)
      • Products were pooled (27/9 , 15:00)
      • No DNA pellet formed after purification and centrifugation. Thus PCR Failed


      Interlab Day 2 protocols were carried out

      • Autoclave falcons + 500 LB broth
      • From each of the 8 plates , inoculate 2 colonies into 5 ml LB + cmp solutions
      • Incubate overnight

      28/9/17

      Pooling PCR 2 (attempt 2)

      • Made reaction mixture of 1000 ul per template , for splitting into 20 * 50 ul reactions
      • PCR reaction mixture (Phu)
        • FP – 50ul
        • RP – 50ul
        • dNTP - 20ul
        • Stock template – 2 ul
        • Buffer - 200 ul
        • Phu pol - 10 ul
        • ddH2O – 668 ul
      • Template stock – 100 ng/ul
      • Ran PCR at 7 PM
      • Ran PCR gel (1% agarose, SB) at 9:30 PM


      Interlab day 3 protocol was carried out

      • Prepare 16 falcons with 12 ml LB+cmp media
      • Take OD600 of each of the above cultures. Calculate the volume required to make the OD600 of the 12 ml culture 0.02 (use the Interlab spreadsheet for that)
      • Aliquot required volumes of each culture into the falcons. Label properly.
      • At t=0 (1130), take out 500 ul from each culture and store on ice.
      • Incubate and shake the cultures at 37 degrees.
      • At t=2 (1330) , t=4 (1530) and t=6 (1730) aliquot 500 ul from the cutltures. Store on ice.

      29/9/17

      Optimizing PCR 5

      • Made reaction mixture of 200 ul per template , for splitting into 8 * 25 ul reactions
      • PCR reaction mixture (Q5)
        • FP – 4ul
        • RP – 4ul
        • Stock template – 80 ul
        • 2x MM - 100ul
        • ddH2O – 12 ul
      • Template stock – 100 ng/ul
      • Ran PCR 6:10 PM
      • Ran products on 1.2% agarose TAE gel 8:20 PM (all temp gave bands)


      Optimizing PCR 6

      • Made reaction mixture of 160 ul per template , for splitting into 8 * 20 ul reactions
      • PCR reaction mixture (Phu)
        • FP – 8ul
        • RP5 – 7ul
        • RP6 - 1ul
        • dNTP - 3.2ul
        • Stock template – 0.4 ul
        • Buffer - 32 ul
        • Phu pol - 1.6 ul
        • ddH2O – 106.8 ul
      • Template stock – 100 ng/ul
      • Ran PCR 6
      • Ran products on 1% agarose SB gel


      Interlab Day 4 protocol was carried out

      • Transfer the collected aliquots into a 96 well plates
      • Take OD600 and fluorescence at 501 (excitation) and 511 (emission)

      2/10/17

      Optimizing PCR 2

      • Made reaction mixture of 160 ul per template , for splitting into 8 * 20 ul reactions
      • PCR reaction mixture (Phu)
        • FP – 8ul
        • RP – 8ul
        • dNTP - 3.2ul
        • Stock template – 60 ul
        • Buffer - 32 ul
        • Phu pol - 1.6 ul
        • ddH2O – 47.2 ul
      • PCR reaction mixture (Control)
        • VF2 – 2ul
        • VR – 2ul
        • dNTP - 0.8ul
        • Stock template – 15 ul
        • Buffer - 8 ul
        • Phu pol - 0.4 ul
        • ddH2O – 11.8 ul
      • Template stock – 100 ng/ul
      • Ran PCR at 6:00 PM
      • Ran products on 1% agarose SB gel at 10:30 PM

      8/10/17

      Optimizing PCR 8

      • Made reaction mixture of 200 ul per template , for splitting into 8 * 25 ul reactions
      • PCR reaction mixture (Phu)
        • FP – 4ul
        • RP – 4ul
        • Stock template – 8 ul
        • 2x MM - 100 ul
        • ddH2O – 84 ul
      • Template stock – 100 ng/ul
      • Ran PCR 11:30 PM
      • Ran products on 1% TAE gel (1:30 AM, 9/10)

      9/10/17

      Pooling PCR 8

      • Made reaction mixture of 1000 ul per template , for splitting into 20 * 50 ul reactions
      • PCR reaction mixture (Phu)
        • FP – 50ul
        • RP – 50ul
        • dNTP - 20ul
        • Stock template – 40 ul
        • Buffer - 200 ul
        • Q5 pol - 10 ul
        • ddH2O – 630 ul
      • Template stock – 100 ng/ul
      • Ran PCR 3:30 AM
      • Ran PCR gel (1% agarose, SB)

      12/10/17

      Pooling PCR 8

      • Made reaction mixture of 500 ul per template , for splitting into 10 * 50 ul reactions
      • PCR reaction mixture (Phu)
        • FP – 25ul
        • RP – 25ul
        • dNTP - 10ul
        • Stock template – 20 ul
        • Buffer - 100 ul
        • Q5 pol - 5 ul
        • ddH2O – 315 ul
      • Template stock – 100 ng/ul
      • Ran PCR at 5:00 PM
      • 1% agarose TAE gel run at 10:00 PM

      Notebook

      Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

      What should this page have?
      • Chronological notes of what your team is doing.
      • Brief descriptions of daily important events.
      • Pictures of your progress.
      • Mention who participated in what task.
      Inspiration

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