Part:BBa_K1298003:Experience

iGEM CoBRA 2014

• DNA Optimization

See BBa_K1298000 (PcChia 1-1) for DNA optimization

• Growing

There are no notes to make on the growing techniques or results.

• Miniprep

First to Third attempt: DNA was not staying inside of the wells, but we did not understand why.

Fourth attempt: Determined that our digested DNA was ‘floating’ out of the wells (and not sinking to the bottom) because we were not dry spinning it enough in this step. The ethanol was still with the DNA after dry spinning, even though it should have been all gone. We concluded that the two dry spins that we were doing was not enough to get rid of the ethanol.

Fifth attempt: Dry spun the tubes four times instead of two. This worked and our digested DNA no longer floated out of the wells while pipetting the DNA inside of the wells.

• Restriction Digest & Gel Electrophoresis

Figure 1: Gel Results of Digestion with Restriction Enzymes on J04500 Plasmids Containing Three Different Chitinase Protein Codes using Two Ladders (June 17th 2014)

#1: (6uL) 1 Kb Ready 1% TBE/Agarose Ladder (the ladder can be found here: http://faculty.pingry.org/aalfano/documents/NEBladderguide.pdf)

#2 - #5: Other Chitinase Digest Results (See BBa_K129004 and/or BBa_K1298005 for more details)

#6: This is the PcChia 1-1 (BBa_K1298000) inside the pSB1C3 backbone with a promoter (BBa_R0010). It was cut with only one restriction enzyme (EcoRI), causing the plasmid to become linearized.

#7: This is the PcChia 1-1 (BBa_K1298000) inside the pSB1C3 backbone with a promoter (Bba_R0010). It was cut with the restriction enzymes EcoRI and PstI, resulting in the coding region to be removed from the pSB1C3 backbone and the promoter (the promoter and backbone are still attached together)

#8: 1 Kb Invitrogen 0.9% Ethidium bromide/Agarose Ladder (the ladder can be found here: http://www.bio.davidson.edu/molecular/Protocols/gels2002/1kbladder.pdf)

#9: (12uL) 1 Kb Ready 1% TBE/Agarose Ladder (the ladder can be found here: http://faculty.pingry.org/aalfano/documents/NEBladderguide.pdf)

• Ligation

Figure 2: Initial Positive Results of Ligation

Both plates were done under the same conditions. This means that we can assume that the colonies from the left-handed plate have the same DNA as the white colonies from the right-handed plate.

• Transformation

Transformation was done using DH5alpha cells, not NEB Top 10-beta cells.

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