SBB09Ntbk-Jennifer Brophy

Jennifer Brophy 00:03, 9 February 2009 (EST)

If possible, make two circularly permutated ompG proteins. One with the backbone opening in loop 2 (aa 59-60) and one in loop 6 (somewhere within aa 220-231) where the residues of the mature protein are not visible in electron density maps, presumably because they are disordered.

to do

Jennifer Brophy 11:59, 9 February 2009 (EST)

Created Construction files for CPG_L2 and CPG_L6. Entered oligos in oligo log

BLAST oligos, enter parts in parts log, look into Nterm and Cterm linker sequences.

Jennifer Brophy 13:19, 18 February 2009 (EST)

Reconstitute oligos. 100uM by adding 10X nmol.

PCR of M10006 (<N.CPG_L2!, regular part, 211bp), A-M10007 (371bp), B-M10007 (343bp), A-M10009 (548bp), B-M10009 (175bp), M10010 (<C.CPG_L6>, regular part, 202bp).

zymo clean-up, PCR A+B for M1007 and M1009.

Jennifer Brophy 21:02, 19 February 2009 (EST)

Did zymo clean up of PCR products (small fragment and regular clean up where appropriate). Diluted some clean ups in to much water so i either redid the PCR or added more template for the A+B reactions. Did PCR A+B of M10007 (694bp) and M10009 (703bp). BUT THESE DIDNT WORK BECAUSE I FORGOT TO DO GEL PURIFICATION TO GET RID OF TEMPLATE DNA.

Jennifer Brophy 18:57, 20 February 2009 (EST)

Did gel purification of M10006,M10007A, M10007B, M10009A (no 9B because i had messed up the zymo clean up yesterday), and M100010. Failure to remove the genomic DNA via gel purification yesterday rendered the PCR of M10007 and M10009 useless.

Gel=ladder, M10006 (211bp), M10007A (371bp), M10007B (341bp), M10009A (548bp), M10010 (202bp).
Re-did PCR of M10009B.

gel purify M10009B. do next round (A+B) PCR. gel purify? start SOEing

Jennifer Brophy 13:07, 23 February 2009 (EST)

Zymo small fragment clean up of M10009B. Gel purify M10009B.

Gel= ladder, M10009B (175bp).
Do A+B PCR to get M10007 (694bp) and M10009 (703bp).

gel= ladder, M10007, M10009. Careful gel purification of M10007, less of a product, mixture of oligos?
Set up PCR of M10008A/B and M10011A/B.

Jennifer Brophy 12:23, 24 February 2009 (EST)

Gel purify M10008A (209bp), M10008B (701bp), M10011A (701bp), M10011B (209bp).

Gel= ladder, M10008A, M10008B, M10011A, M10011B.
Set up PCR for M10008 and M10011.

to do

Jennifer Brophy 13:27, 25 February 2009 (EST)

Gel purify M10008 (892 bp) and M10011 (892bp).

Gel= ladder, M10008, M10011.
Did Eco/Bam digest and ligation into pBca9495KC. Plated on KC plates.

to do

Jennifer Brophy 13:29, 27 February 2009 (EST)

Innoculate two tubes of M10011.
Re-do ligation of M10008 because no colonies grew. Redo plating.

pick M10008 colonies. 
miniprep colonies. Do restriction mapping: (parts >250 bp= EcoRI/BamHI) 
 6.5uL water
 1 uL NEB2
 2 uL plasmid
 0.5 uL EcoRI
 0.5 uL BamHI or XhoI
30 min at 37C 
run gel
turn in for sequencing
 

Jennifer Brophy 01:18, 1 March 2009 (EST)

Picked M10008 colonies. Plate looked very good. Many colonies grew.

to do

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Notes

to do