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

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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

electrophoresis result

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.


Lock & Key By Yu Tao

Transformatiion Test of the Newly Prepared Competent Cells (Competent Cells I)

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.
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