Samantha Liang Notebook
From 2007.igem.org
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*All the other PCRs looked fine on a gel | *All the other PCRs looked fine on a gel | ||
*Cleaned up Digested and inserted all PCR products into Biobrick form (I716210 through I716113) | *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 | + | *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 | *Picked colonies from pBca9145-I716207-1 and pBca9145-I716208-1 | ||
Revision as of 22:08, 7 June 2007
My Construction Files
My Sequencing Files
My Biobricks
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
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
