Innoculate Ben’s mammalian backbones with dimerization domains and Ben’s phiC-31, TP901-1, ofr7 and gp3 plasmids
May 29th 2015
Cell stock creation from frozen plasmids
Miniprepped the plasmids we grew up and cell stocked for iGEM cell stocks
Using the nanodrop fluorospectrometer
Week Two - June 1st to June 5th
June 1st 2015
Digested Ben's backbones
PCR set-up
Made an agarose gel to separate the products of the PCR
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
Mini-prepped the pS-5 and pS-18 cell stocks that were made yesterday.
Measured the concentrations of DNA from the purified digestion results and purified gel results.
Digested pS-5 because it is a backbone. pS-18 is not a backbone that is why it was not re-digested.
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.
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.
Gel purified
Digesting Our Two Integrases with Enzymes in Order to Ligate (the pS-14 phi 31 integrases into pS-1:12 backbones)
PCR Purification of Our Restriction Results to Get the Integrase Inserts
Ligation
June 3rd 2015
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
Picked colonies
Emailed former BU students who developed a light stimulation device
June 4th 2015
Took colonies out of incubator, decanted media
Miniprepped from deep wells
Test cut plasmids
Ran gel electrophoresis
Week Three - June 8th to June 12th
June 8th 2015
Repicked colonies for bad test cuts
200, 201, 204, 205, and 208
Created new deep well plates with LB + Carb
Picked three new colonies from each of these plates
Incubated reference plate and deep well plate overnight
Saving a Gel (There was an incorrect number of wells in a gel we made so: )
We tore up the gel and put it into the iGEM gel erlemyer flask
Microwaved the 100mL of gel for one minute until it was melted
We re-added the ethidium bromide
We re-poured the gel
June 9th 2015
Redid test cut of bad test cuts from yesterday
Miniprep colonies
Digest plasmid and incubate for 30 minutes
Run gel to compare current digested bands to how long they should be (found by looking at initial sequence)
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
Re-did the gel again, got the same results
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
Jeff re-made the PCR solutions
We ran the PCR with the correct iGEM settings
Ran a gel with the PCR results
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
Ran a gel with the new PCR products; we'll call this gel #2
Cut out the fragments we identified as the proper inserts
Purified them
Digested the fragments
Purified the digested fragments
June 11th 2015
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.
Today, we redid the test cuts for pRECs 195, 196, 197, 198, 199, 203 and 207
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)
Digested the backbones. Instructions from Ben’s ABA and rapamyacin backbones in order to get the inserts we want to send for sequencing
Abha and Jeff redid the ligation for pREC 200, 201, 204, 205, 208.
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
After digesting Ben’s Inserts, we are going to run a gel and purify the bands that correlate to the insert
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.
Ben’s inserts digested really well and we now we are going to ligase the purified bands (inserts) into the backbones
June 12th 2015
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.
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.
Shaheer harvested the colonies from his transformed plates, as described by the Deep Well Miniprep procedure.
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.
Ben analyzed the results and hypothesized that our pS-9”12 backbones were digested incorrectly.
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.
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.
Jeff and Kate digested the backbones
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.
