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): 1,4,5 PlacI-I741051 @ EcoRI/PstI 2,3,4 J61003 @ EcoRI/PstI 7    I741051 @ EcoRI/PstI
 * From left to right:

=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.

=Lock & Key By Yu Tao=

Transformation Result

 * Number of colonies:
 * 1) Previous competent cells: more than 2000 clones.
 * 2) Newly prepared competent cells: fewer than 1000 clones.
 * Comments: I think the efficiency of the new cells is a little bit lower and I decide to make some more competent cells tomorrow.

J01010 and J01008 (both by PCR) Digestion Product Purification

 * Both of the fragments are digested from yesterday.
 * use Transgen EasyPure PCR Purification Kit / Quick Gel Extraction Kit.
 * 50uL per tube after purflication, one tube, respectively.

Electrophorsis Result

 * from left to right:
 * 1) J01010 @ XbaI/PstI (2uL)
 * 2) J01008 @ XbaI/PstI (2uL)
 * 3) DL plus 2000 marker
 * 4) J01010 PCR product (2uL)
 * 5) J01010 PCR product (2uL)
 * 6) J01008 PCR product (2uL)
 * 7) DL plus 2000 marker
 * 8) marker

Ligation: R0010<-J01008 and R0040<-J01010

 * Ligate the J01008 fragment and R0010 vector, also the J01010 fragment and R0040 vector.
 * I use the previous digested R0040 @ PstI/SpeI and R0010 @ PstI/SpeI.

Electrophorsis Result

 * from left to right:
 * 1) R0040 @ PstI/SpeI (Use PCR purification kit)
 * 2) R0010 @ PstI/SpeI (Use PCR purification kit)
 * 3) DL plus 2000 marker
 * 4) marker


 * Comments: The R0040 @ PstI/SpeI seems degraded
 * Ligation system contains:

8 µl      J01008 fragment / 6.5 µl       J01010 fragment 0.5 µl    R0010 vector / 2 µl     R0040 vector 0.5 µl    Super T4-Ligase 1 µl      10 X ligation buffer -- 10 µl     Total


 * The negative control group contains no fragment but the same volume ddH2O instead.
 * 10min at 16℃

Transformation: the new ligation product above

 * Transform each 10 µl ligation system into 100 µl DH5α competent cells.
 * Culture R0010<-J01010 cells at Amp+ and R0040<-J01008 LB plate for 12 hours.
 * Result to be seen tomorrow.