Difference between revisions of "Team:BostonU/Notebook"

Notebook

May

Week One - May 28th to May 29th

• May 28th 2015
  1. Made broth and agar
  2. Plated the agar
  3. Aliquot 50mL LB media tubes
  4. Innoculate Ben’s mammalian backbones with dimerization domains and Ben’s phiC-31, TP901-1, ofr7 and gp3 plasmids
• May 29th 2015
  1. Cell stock creation from frozen plasmids
  2. Miniprepped the plasmids we grew up and cell stocked for iGEM cell stocks
  3. Using the nanodrop fluorospectrometer

Week Two - June 1st to June 5th

• June 1st 2015
  1. Digested Ben's backbones
  2. PCR set-up
  3. Made an agarose gel to separate the products of the PCR
  4. Made more pS-5 and pS-18 cell stocks (Our pS-5 and pS-18 cell stocks gave us a very low yield when their concentrations were tested with a nanodropper).
• June 2nd 2015
  1. Mini-prepped the pS-5 and pS-18 cell stocks that were made yesterday.
  2. Measured the concentrations of DNA from the purified digestion results and purified gel results.
  3. Digested pS-5 because it is a backbone. pS-18 is not a backbone that is why it was not re-digested.
  4. Ran the PCR for pS-14 and 10 different primers for a second time, the first time we did this was yesterday but the test yielded strange results in that our bands were thick, clouded, and our ladder was relatively useless because it did not run for a long enough period of time.
  5. Ran the gel for the results from today’s PCR in order to see if we can get better results than yesterday and we did.
  6. Gel purified
  7. Digesting Our Two Integrases with Enzymes in Order to Ligate (the pS-14 phi 31 integrases into pS-1:12 backbones)
  8. PCR Purification of Our Restriction Results to Get the Integrase Inserts
  9. Ligation
• June 3rd 2015
  1. Checked out the colonies and Ben found that our pS-9 and pS-11 had very low yields when checked against their respective negative control colonies
  2. Picked colonies
  3. Emailed former BU students who developed a light stimulation device
• June 4th 2015
  1. Took colonies out of incubator, decanted media
  2. Miniprepped from deep wells
  3. Test cut plasmids
  4. Ran gel electrophoresis

Week Three - June 8th to June 12th

• June 8th 2015
  1. Repicked colonies for bad test cuts
     1. 200, 201, 204, 205, and 208
     2. Created new deep well plates with LB + Carb
     3. Picked three new colonies from each of these plates
     4. Incubated reference plate and deep well plate overnight
  2. Saving a Gel (There was an incorrect number of wells in a gel we made so: )
     1. We tore up the gel and put it into the iGEM gel erlemyer flask
     2. Microwaved the 100mL of gel for one minute until it was melted
     3. We re-added the ethidium bromide
     4. We re-poured the gel
• June 9th 2015
  1. Redid test cut of bad test cuts from yesterday
     1. Miniprep colonies
     2. Digest plasmid and incubate for 30 minutes
     3. Run gel to compare current digested bands to how long they should be (found by looking at initial sequence)
  2. Notice that maybe inserts 4, 5 and 8 are not properly ligated, contaminated or in general not properly prepared so plan to redo PCR of those insert fragments and start from there
  3. Re-did the gel again, got the same results
  4. Tried to PCR prep 4, 5 and 8 but the settings were incorrect so we threw away the samples and resolved to start again tomorrow.
• June 10th 2015
  1. Jeff re-made the PCR solutions
  2. We ran the PCR with the correct iGEM settings
  3. Ran a gel with the PCR results
  4. We decided to re-do the PCR but use half the amount of primmers in pREC 205 and 208 because there may be a primer dimer band showing up; we also decided to double the extension time because pREC 204 looked like it wasn't getting enough time to extend
  5. Ran a gel with the new PCR products; we'll call this gel #2
  6. Cut out the fragments we identified as the proper inserts
  7. Purified them
  8. Digested the fragments
  9. Purified the digested fragments
• June 11th 2015
  1. Our previous ligation did not work, it had grown no colonies when we got into lab this morning and we did not make a negative plate.
  2. Today, we redid the test cuts for pRECs 195, 196, 197, 198, 199, 203 and 207
  3. Today we are also going to send DNA for sequencing, based on gel results, we have decided to send pREC 193 (2), 194 (1,3), 202 (1,2), 206 (2,3)
  4. Digested the backbones. Instructions from Ben’s ABA and rapamyacin backbones in order to get the inserts we want to send for sequencing
  5. Abha and Jeff redid the ligation for pREC 200, 201, 204, 205, 208.
  6. Abha and Jeff did the transformation by smearing 50 microliters of this mixture: 50 microliters Top 10 Cells, 50 microliters KCM, 10 microliters ligation; placed in incubator at noon
  7. After digesting Ben’s Inserts, we are going to run a gel and purify the bands that correlate to the insert
  8. The results of my test cuts turned out pretty much the same as the June 5th gel so we are not going to send any additional pRECs for sequencing.
  9. Ben’s inserts digested really well and we now we are going to ligase the purified bands (inserts) into the backbones
• June 12th 2015
  1. Today we checked Abha and Jeff transformations. We found no growth on the plates except for the negatives, which had quite a bit. This means that most likely we did not digest the backbones properly or we did not perform the ligation correctly.
  2. Shaheer’s plates with Ben’s inserts and the iGEM team’s backbones did yeild positive results. Additionally, Shaheer’s negative controls did not grow any colonies, so we can be confident that the colonies obtained are transformed colonies.
  3. Shaheer harvested the colonies from his transformed plates, as described by the Deep Well Miniprep procedure.
  4. We got back the results of the pRECs Ben sent for sequencing yesterday and have yet to cross examine them with their expected sequences. All of the pRECs chosen, with the exception of pREC 206 (3), yeilded at least one “Good” sequence.
  5. Ben analyzed the results and hypothesized that our pS-9”12 backbones were digested incorrectly.
  6. We are going to test cut the backbones to verify their sequences by digesting each backbone with the opposite enzyme with which we are going to ligate it.
  7. Because of all of the vectors looked exactly as expected, we suspect the switch up Ben identified from the sequencing occurred at some later point.
  8. Jeff and Kate digested the backbones
  9. Kate did the digestion with two mastermixes, 240 microliters water, 40 microliters buffer, 12 microliters enzyme 1 and 12 microliters enzyme 2. Then she loaded 38 microliters mastermix into the corresponding cryotubes, then added 12 microliters DNA to the corresponding cryotubes.
• June 13 2015
1. Centrifuged deep wells at 3500 rpm for 6 min
2. Removed supernatant
3. Sttored in -20 degrees celsius fridge

Week Four - June 15th to June 18th

• June 15th 2015
  1. Abha and Jeff miniprepped their ligated plasmids from the deep wells
  2. Then they test cut them; the results did not turn as desired
• June 16th 2015
  1. The colonies looked good so we picked three from each pREC colony for pRECs 193-208.
  2. Abha filled a deep well with 2 mL LB brother + carb for growing up cell colonies
• June 17th 2015
  1. We centrifuged the deep wells at maximum speed for 6 minutes.
  2. We miniprepped Ben’s inserts and iGEM backbones from the deep wells.
  3. Abha and John started the PCR of our new prRECs.
  4. We test cutted the miniprepped plasmids and found that pRECs 193-200 turned out really well but the pREC 201-208 did not show up. We hypothesized that there is something wrong with the enzymes EcoRI or Kpn1 or buffer.
  5. We test cutted pRECs 201-208 again, and found the same results, which were that a backbone appeared in the gel, but no insert was digested out.
  6. Abha and Jeff ran a gel for the prRECs that they PCRed and found that their results were not promising.
• June 18th 2015
  1. Test cutting Divya’s PO-004 plasmids to see if our EcoRI or Kpn1 or Cutsmart buffer is bad.
  2. Ben said this morning that our sequencing results from pS-9 and pS-11 are very promising!
  3. Our test cut did not work. This means that we do not know whether it is our enzymes or our buffer that is faulty.
  4. PCR of prREC1-36 (1-18 for TP901 and 19-36 for PhiC31)
  5. Gel purified prREC 1-36 (many did not show the proper band size)
  6. Made new carb+agar plates

Week Five - June 22nd to June 26th

• June 22nd 2015
  1. Test cutting pS-10 and pS-12 pRECs 201-208 with Mlul and AccIII because we may have a bad enzyme in the EcoRI/Kpnl digests of pREC 201-208
  2. Our gel returned the expected results from our test cut. This means that our issue probably lies in the enzymes or buffer and not in the plasmids themselves.
  3. Nanodropped purified prRECS 1-36 and many had low yield
  4. Redid PCR on 17 prRECS (most bands aligned the expected size!)
  5. Repurified redone PCRs
  6. Sent 23 tubes for sequencing
     1. pREC 193-196 using BHW100 reverse primer (this is redoing the reverse CIBN sequencing from 06/17/15 because the reverse primer was too far down to show anything in the actual PhiC31 region)
     2. pREC 197 (3 colonies) using BPO0038 forward primer (for some reason these were not sent in the 06/17/15 sequencing; reverse was not sent because we do not have the proper CRY2 reverse primer)
     3. pREC 201-204 using BPO0038 forward primer and BHW98 reverse primer
     4. pREC 205-209 using BHW99 forward primer and BHW100 reverse primer
  7. Ordering from Quintera:
     1. label plasmid files with pREC # and primer used
     2. open recent orders
     3. download then extract the zipfile; copy extracted files into dropbox
     4. open .seq file in Ape: compare to pREC sequence map in the dropbox
     5. Tools -> align two sequences (align the quintara sequence with the pREC map)
     6. Mismatches towards the end are okay because there is a lot of noise towards the end of sequencing (you can see the progression in the .api quintara files)
     7. Reading and sequence verifying the alignment data: wrong primer would give you a lot of red in the results where there were mismatches; good results would show matching in the backbone and part of the phiC31 domain (verifies correct plasmid backbone and insert ligated)
• June 23rd 2015
  1. Split into Team A and B, Team B is in charge of Ben’s primers and the Cas9 splits.
  2. We read the results of the sequencing from Monday about pRECs 197-208 and did not get good results - Divya hypothesizes that this is because there is something wrong with our Primers or on Quintara’s end. She said she was having trouble with her sequencing as well and that is why she thinks it is a sequencing problem and not a plasmid/ligation problem.
• June 24th 2015
  1. We are going to re-send pRECs 197-208 for sequencing

Week Six - June 29th to July 3rd

• June 29th 2015
  1. The prRECs 37-54 came in. We added the micromolar amount of autoclaved deionized water specified on the IDT prREC order sheets.
  2. Then we labeled microcentrifuges for each prREC and made a working stock by adding 90 microliters deionized autoclaved water to the microcentrifuge tubes so that we could use only one tip. Then we added 10 micromolars of each respective prREC to each microcentrifuge tube so that we have a 10 micromolar working stock now.
• June 30th 2015
  1. Shaheer re-digested the backbones pS 1 to pS 4, and digested backbones from pS 1:4 stock. He gel purified the samples and these are what we will use in our ligation later today.
  2. PCR of prRECs 37-54
  3. Attempt #1: Jeff and John ligated the pRECs
• July 1st 2015
  1. Attempt #2: Jeff re-transformed the ligations from yesterday
• July 2nd 2015
  1. Attempt #3: Shaheer re-ligated the pRECS that did not work from Attempt #1 and #2
• July 3rd 2015<
  1. Attempt #4: Jeff and Shaheer re-ligated the pRECs that did not work from Attempt #1, #2, and #3
• July 4th 2015
  1. Jeff picked the colonies from ligation attempt #4 and set them up for inoculation
• July 5th 2015
  1. Shaheer mini-prepped the deep wells from ligation attempt #4.

Week Seven - July 6th to July 10th