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

Line 292: Line 292:
 
<li>Transfer the collected aliquots into a 96 well plates
 
<li>Transfer the collected aliquots into a 96 well plates
 
<li>Take OD600 and fluorescence at 501 (excitation) and 511 (emission)
 
<li>Take OD600 and fluorescence at 501 (excitation) and 511 (emission)
−
         </ul
+
         </ul>
  
  

Revision as of 07:07, 1 November 2017

Preparation

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

You can see what others teams have done to organize their notes: