IGEM:Peking/2007/Count-Conjugation-Notebook/2007-8-11

Tandem-OriT by Qu Mingzhi

OUR FIRST Biobricks !!

• F-OriT-J23066-OriT pSB1A2 now named as I741051!
• waiting for sequencing to get the final detail.

re-do: PlacI -> I741051

• basic experiment condition :IGEM:Peking/2007/Count-Conjugation-Notebook/2007-8-10
• we changed "fast T4 ligase" ligation time : from 20min ->10 min @16℃
• digesting for longer time.
• use both "fast T4 ligase" & normal T4 ligase".

I741051 vector

• From left to right:

1. Maker (DL2000 Plus)
2. I741051 vector (after Gel extraction, EcoRI/XbaI)

R0010 before gel extraction

• From left to right:

1. R0010 -1 @ EcoRI/SpeI
2. R0010 -2 @ EcoRI/SpeI
3. Maker (DL2000 Plus)

R0010 after gel extraction

1. R0010
2. Maker (DL2000 Plus)

after ligation & mini-prep

NEXT DAY(8-12):

• From left to right:

  1,4,5 PlacI-I741051 @ EcoRI/PstI
  2,3,4 J61003  @ EcoRI/PstI
  7     I741051 @ EcoRI/PstI

oriT Knock Out

• By Xu Anting

Transformation of pKO3-oriT-deleted plasmid into DH5a

• Result: negative controls have an approximately number of clones in comparing with ligation products. It seems that our DH5a have been contaminated.

Ligation Products Verification

• By Colony PCR
• Picked colonies and grew them in an additional Cm Petri-dish, in 37 centrigrade, overnight.