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From 2014.igem.org

• Week 1

  • picked single MG1655 and pTRC99A strains into liquid culture, incubated overnight (pTRC99A in LB+Amp)
  • miniprepped pTRC, O/N culture of MG1655
  • MG1655 did not grow on antibiotic plates, so stocks are good!
  • prepped DNA for transformation – pCas9 and RFP reporter
  • made MG1655 electrocompetent – electroporate and plated transformants, incubated overnight

• Week 2

  • transformation of RFP, pCas9, GFP, fmt into NEB5alpha
  • transformants plated and incubated overnight
  • then inoculated into liquid culture, incubated overnight
  • overnight culture of MG1655 and pTRC99A
  • Miniprepped RFP and pTRC99A
  • tested restriction enzymes EcoRI, Xbal, SpeI, Pstl on miniprep and ran on gel w/ uncut as control
  • cut plasmids were too faint to see – need more concentrated DNA in preps!
  • O/N cultures of RFP, pCas9, GFP from transformation
  • miniprep of O/N cultures of transformants and pTRC99A

• Week 3

  • miniprep again, greater volume (3mL) for RFP, GFP, Cas9, pTRC99A + nanodrop
  • digest product (w SpeI, EcoRI, cutsmart) and run on gel
  • O/N culture from plate of extra biobrick, RFP, MG1655
  • start RFP and MG1655 liquid cultures for the RFP spectrum measurements in the afternoon
  • RFP grew slowly and was transformed into NEB5alpha instead of MG1655
  • digest 8 samples from yesterday’s miniprep of 8 biobrick cultures, load and run gel of digest
  • oligos and gblocks arrived
  • did PCR of pCas9 and pTRC99A chunks
  • Started MG1655 liquid culture for making chemically competent
  • make chemically competent cells, transform RFP into them, PCR purification, run on gel

• Week 4

  • miniprepped pCas9 + pTRC99A (w 7mL of cells)
  • did gel extraction – cut out pCas9 + pTRC digests – PCR both as well as nanodrop
  • Mon/Tues = PCR w mastermix, Wed = make comp MG1655 and heatshock RFP, Thurs/Fri = confirm RFP and Marafini protocol/golden-gate assembly
  • PCR from the Friday before did not work
  • so do lots of minipreps from 10mL cultures and start over, use larger volumes for digests
  DNA eluted into collection tube but saw salt contamination when nanodropped therefore redo miniprep…
  • miniprepped pTRC and pCas9 and then nanodropped – results were good so created gel for next day, as well as a digestion
  • digested/ran the gel/extracted, will nanodrop gel extraction – if worked, try PCR again

• Week 5

  • Wed. minipreps were good (however possibly from just high free nucleotides), gel extraction failed (no DNA at the end and a lot of salt contamination) – so just PCR from miniprepped plasmids
  • digest and run gel of digest at the same time!
  • PCR of pTRC and pCas9 – have also been digested
  • ran gel! 5 part sample, 1 part loading dye
  • if PCR worked, digest PCR product w DpnI – got nothing for pCas9 though on the gel (to be re-PCR-ed), but bright bands for pTRC
  • competent MG1655 also done
  • PCR of miniprep C1 #1 of cas9… did 10 x 10uL reaction using gradient temps in 58-68 range
  • run on gel to see if PCR worked
  • PCR gel showed 2 weak bands – either not enough DNA or PCR failed
  • heat shock transformation for MG1655 w RFP plasmid but very few colonies on CM plate

• Week 6

  • PCR product (???) run on gel, pick most successful PCR
  • DpnI digest following protocol in NEB Gibson manual Transform RFP & MG1655, O/N cultures
  • pCas9PCR product visible on gel
  • DpnI digest + PCR cleanup of digest (ended up not having enough DNA)
  • plating of transformed RFP into DH5a
  • processed PCR – ran on gel, DpnI digest also ran on gel, PCR cleanup, nanodrop (worked well)
  • needed to transform pCas9+spacer plasmid & RFP DH5a

• Week 7/8

  • transform RFP & BL21(DE3)
  • PCR of pCas9, pTRC99A
  • Gibson – pick 8 colonies, make O/N colonies, try to PCR form reaction to get 4-fragment superchunk
  • Maraffini – use Anderson high for 4 fragment (phosphorylation + annealing of spacers if we get a lot of DNA), otherwise using Aug 18 miniprep do a BsaI digest
  • got colonies from Gibson
  • 3 things in parallel:
  • miniprepped plasmids from Gibson transformants from O/N liquid cultures (need to be sent for sequencing) , plasmids digested w E+P and to be run on gel
  • making competent MG1655 – transform RFP into it
  • tried BsaI digestion and gel extraction on Stanford-brown pCas9 (for Marafini/Golden-gate), does not seem to work even w large volumes
  • look at plates from transformation, run E+P digests on gel – if the digests look wrong size, do colony PCR on rest of Gibson colonies on plates
  • use fwd/rev primers for cas9 and p!TRC chunks
  • if correct, also PCR the 3-fragment superchunk from the miniprep plasmid by using Cas9 fwd with p!TRC rev primers
  • try a gel-extraction protocol…

• Week 9

  • Main thing – GIBSON ASSEMBLY
  • screened 8 colonies – miniprepped, EcoRI + PstI digest & wrong product, non-specific assembly
  • extend gblocks (Anderson high + tracr) so more overlap, try Gibson again
  • did digests of plasmids w one restriction enzyme instead of double digest + run gel (for less confusing results) – used miniprep #1-4
  • made O/N cultures from first Gibson reaction #9-15 – miniprepped and streak out plates
  • minipreps #5-8 from earlier digested w EcoRI and PstO (separately), run on gel
  • take 2 minipreps, do 3 reactions on each – EcoRI only, PstI only, both EcoRI + PstI

• Week 10

  • miniprep out RFP plasmid to be used for putting biobricks into pSB1C3, will want to screen for non-red colonies at the end of the process
  • unable to do digest because nanoprep of all 3 minipreps was too low
  • primers arrived – ran PCR w new primers
  • also digested RPF
  • run digests on gel (1/2 thickness)
  • ran the original gblocks w extended gblocks – not enough of original gblocks to show up on gel
  • combined PCR reactions of tracr gblocks into one, same with Anderson High PCR products
  • purified both
  • PCR purified Cas9 array from a PCR in August
  • purified p!TRC chunk found
  • all 4 to be used for Gibson assembly!
  • glycerol stocked 3 tubes of colonies from first Gibson attempt (#3, 4, 7)
  • transformed Gibson in NEB5alpha, ½ of each reaction tube into a total of 4 NEB5alpha – 4 tubes plated onto 2 plates each = total 8 plates incubated
  • on Friday, found colonies from the Gibson assembly and did colony PCR
  • colony PCR primers were designed to cover the seams where the 4 chunks joined
  • mixed the template DNA and biobrick primers for the Cas9-array chunk, but did not add the master mix yet (done later)

• Week 11

  • ran Gibson colony PCR on gel – colonies #3 + 6 have correct bands, started O/N cultures of them w cell suspensions from colony PCR
  • miniprep and glycerol stock them (low copy plasmid – do 3uL miniprep)
  • PCR of cas9 chunk – run on gel, see if band is at 4595
  • using RFP miniprep from earlier, digest w EcoRI and PstI
  • linearised plasmid digested same way (if DNA dissolved – was left sitting overnight to dissolve)
  • nothing on gel at correct size…. only used 1uL of DNA for gel though
  • digest ~1000ng of Gibson miniprep from 2 colonies w EcoRI and PstI, run on gel
  • run 5uL of tube labelled C6*
  • cas9-array chunk biobrick extension PCR barely worked – not enough DNA after PCR purification but tracr chunk biobrick PCR worked
  • digested/ligated into pSB1C3, transformed into competent cells
  • Gibson colonly #3 didn’t assemble correctly, only produced 1 band from E+P digestion
  • transformed ligation into NEB5alpha – after 2 hour (actual = 10 min) incubation in SOB, pipetted into 2mL LB Cm and grew overnight
  • miniprepped to see if it grew and packed it up for shipping!