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)

Peking 2007-8-11 I741051 vector.jpg

R0010 before gel extraction

  • From left to right:
  1. R0010 -1 @ EcoRI/SpeI
  2. R0010 -2 @ EcoRI/SpeI
  3. Maker (DL2000 Plus)

Peking 2007 8-11 R0010-5+EcoR1+Spe1.jpg

R0010 after gel extraction

  1. R0010
  2. Maker (DL2000 Plus)

Peking 2007-7-11 PlacI after extraction.jpg

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

Peking 2007-8-12 1 5 6J61003E-p 2 3 4-plac-I74051E-P 8-I74051E-P.jpg

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

Example.jpg

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

Example.jpg

  • 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.