Samantha Liang Notebook


Revision as of 21:55, 14 June 2007 by Samanthaliang (Talk | contribs)

My Construction Files
My Sequencing Files
My Biobricks

Samanthaliang 15:28, 14 June 2007 (EDT)

  • sequencing for the GTG start Cre was same as before - still has an ATG start - I can't really figure out what is going wrong here. I definitely used dpnI to digest the parent vector because I remember having to go upstairs to the Keasling lab to get hooked up with that enzyme since we were out of it downstairs. Also, both times I did it, there were a lot of colonies on the plate, and there would have been none if it were the parent vector since I used dpnI. It must be some problem with the PCR then - is it self-correcting? I double checked the oligo and it definitely has a GTG start. I guess I'll try to make it one more time, but if it doesn't work this time then I might have to try Quick Change if it's worth having this part.
  • Grow up colonies for cassettes+toxins (216-223)- the plates are alright. except for the ones with BamHI, those plates only had one colony each. weird. maybe a problem with the digest since both of them are bad? Will re-ligate and transform today hoping for an easy fix. If the sequencing comes out bad though eventually, I will have to redo the subcloning for 218 and 219 from the start.
  • Make the ape files for cassette+toxins
  • Read that locks and keys article
  • Subclone Cre (GTG) over.

Samanthaliang 18:21, 13 June 2007 (EDT)

  • miniprepped 212s - sequenced the clones that came from the plate with more colonies (put cultures in fridge)
  • subcloned all the cassettes+toxins (216-223) because sequencing came back good on the cassettes for the clone 2s - 8 different new plasmids!
  • put teeth on the centrifuge

Samanthaliang 15:26, 12 June 2007 (EDT)

  • grew up 212 clones
  • miniprepped 214 and 215 clones and sent them out for sequencing
  • clone saved
  • sequencing came in and it looks like we have a good clone for every toxin!
    • ceaB (209) - clone 2
    • BamHI (210)- clone 2
    • barnase (211)- clone 1
    • BglII - already a miniprep Bca1147
  • still missing Cre with a GTG start - is being grown up today
  • Cre with a TTG start (213) clone 2 is perfect
  • Made construction files for cassette+toxin
  • Now in charge of the google spreadsheets - have to invite people and what not

For tomorrow: Once the cassettes have been confirmed for sure, can digest those and the toxins and ligate.

Samanthaliang 19:24, 11 June 2007 (EDT)

  • grew up 214 and 215 clones
  • Sequenced 209 (clone 1&2), 211 (clone 1&2), and 213 (clone 2) again with G01001 (backwards) because the genes were too big to confirm for sure that they were right, 210 was perfect the first time
  • Redid the PCR for 212 (Cre-GTG) because both sequences came back with the ATG start codon still
  • Subcloned 212
  • Clone saved and recorded on the new Google Spreadsheet
  • emailed china team that wanted our oriT knockouts from last year - told them to look in the registry again

Samanthaliang 20:00, 9 June 2007 (EDT)

  • Miniprepped 209, 210, 211, 212, 213 and sent them out for sequencing
  • Sequencing came in for 207 and 208 - should use clone 1 for both.

Will try to come in on Sunday too to grow up the 214 and 215 cultures - i moved the plates to the fridge for now.

Samanthaliang 17:58, 8 June 2007 (EDT)

  • Made a ridiculously large batch of DH10B competent cells
  • Miniprepped 207 and 208 clones, clone saved them, and sent them out for sequencing
  • Used those preps to subclone I716214 and I716215 (two versions of each)
  • Grew up cultures of 209, 210, 211, 212, 213

Samanthaliang 17:58, 7 June 2007 (EDT)

  • Chris gave me the wrong template for Cre and I didn't notice until today so I redid those 2 PCRs
  • All the other PCRs looked fine on a gel
  • Cleaned up Digested and inserted all PCR products into Biobrick form (I716210 through I716113)
  • Got sequencing data back and only one of the [rbs][atg] ones came out good, but the [lox][terms]s all came out good - just forget about the [rbs][atg] ones for now. More detail - the ones that failed also had really tiny colonies and were in Bca1101 form with a promoter in front, so it might be translating some nonsense that is toxic to the e.coli - but since I have one of the rbs's already I'm just going to proceed with that
  • Picked colonies from pBca9145-I716207-1 and pBca9145-I716208-1
  • Austin grew up 10ml of DH10B for me so I can make competent cells tomorrow

Samanthaliang 17:31, 6 June 2007 (EDT)

  • miniprepped 201 through 206 and sent them out for sequencing
  • put the clones on "clone saver" paper
  • subcloned 207 and 208 with all clones (have 2 versions of each just in case)
  • PCRed ceaB, BamHI, Barnase, Cre with both alternate stop codons

Samanthaliang 19:20, 5 June 2007 (EDT)

  • Grew up 2 colonies each of I716201 through I716206
  • Made a lot of construction files and entered things into the registry
  • My oligos were ordered today!
  • Demonstrated liquid media making and registry entering
  • Now will also make an RFP variant as a reporter on how well Cre is working

To do tomorrow:

  • subclone 207 and 208
  • Run PCRs if they come in the afternoon
  • Send things out for sequencing and make -80s
  • Fill out and sent those survey forms

Samanthaliang 15:29, 4 June 2007 (EDT)

  • Oligos to make biobricks of toxins are not yet in - maybe Wednesday or Thursday?

For cassette between Cre and toxin: [rbs][ATG][Lox][Term][Lox]

  • Made [rbs][ATG] variants with different ribosome binding sites
       I716201 is Bca1106A.Bca1112 
I716202 is Bca1106B.Bca1112
I716203 is Bca1128A.Bca1112
I716204 is Bca1090.Bca1112
  • Made [Lox][Term] variants with 2 different terminators (will probably use Bca1092 version)
     I716205 is Bca1114.Bca1124 
I716206 is Bca1114.Bca1092
  • Tomorrow will make [Lox][Term][Lox] part
  • Then will put the rbsATG together with LoxTermLox

eek so many construction files and ape files to make.

to do