Samantha Liang Notebook
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Revision as of 19:26, 12 June 2007 by Samanthaliang (Talk | contribs)
My Construction Files
My Sequencing Files
My Biobricks
Samanthaliang 15:26, 12 June 2007 (EDT)Samanthaliang 19:24, 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