IGEM:Peking/2007/Count-Conjugation-Notebook/2007-7-28

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Contents

oriT Knock Out

  • By Xu Anting
The transformation of ligation products finished, with incubating the resulting DH5a in LB Amp X-gal, 37 centigrade. Hoping to get results tomorrow.

Lock & Key By Yu Tao and Zheng Qinsi

Mini-prep: R0010, J23066 and B0015

  • Using Transgen mini plasmid purification kit.
  • 50uL per tube after purification, 2 tubes per type of plasmids.

Mini-prep single digesting test result

  • Digesting all the plasmids with EcoRI.
  • Each digestion system contains:
1 µl       10*H buffer
0.25 µl    EcoRI
3 µl       Plasmid
6 µl       ddH20
--------------------------
10 µl      Total
  • 37℃ culutre for 3 hours.
  • from left to right:
  1. $$$ @ EcoRI
  2. marker (DL2000 Plus)

Image:Example.jpg

Mini-prep double digesting test result

  • Samples stored at -20℃ last night.
  • from left to right:
  1. R0010<-J23066-1 @ EcoRI
  2. R0010<-J23066-1 @ XbaI/PstI
  3. R0010<-J23066-2 @ EcoRI
  4. R0010<-J23066-2 @ XbaI/PstI
  5. R0010<-J23066-3 @ EcoRI
  6. R0010<-J23066-3 @ XbaI/PstI
  7. R0010 @ PstI/SpeI
  8. J23066 @ PstI/XbaI
  9. marker (DL2000 Plus)

Image:Example.jpg

  • Still there are some unknown bands shown up in R0010 and B0015 plasmids. I suggest a re-mini-prep again.

B0015, E0040(pSB3K3) and R0040 Double Digestion Product Purification

  • The digestion was under way yesterday.
  • use Transgen EasyPure PCR Purification Kit for E0040(pSB3K3) and Quick Gel Extraction Kit for B0015.
  • 30uL per tube after purflication, one tube, respectively.

Electrophorsis Result

  • from left to right:
  1. marker

Image:Example.jpg

Ligation: E0040(pSB3K3)<-B0015

  • Ligate the B0015 fragment and E0040(pS3K3) vector
  • Ligation system contains:
7 µl       B0015 fragment
1 µl       E0040(pSB3K3) vector
1 µl       T4-Ligase
1 µl       10 X ligation buffer
0 µl       ddH20
--------------------------
10 µl      Total
  • The negative control group contains no fragment but 7uL ddH2O instead.
  • 4℃ overnight.

Transformation Result: J23078(pEASY-T2)

  • There are colonies both in the experimental plate and the negative control plate.
  • Both plates are of no differences in the number of colonies.
  • Number of colonies: 5 in the experimental plate and 2 in the negative control plate.
  • Select 2 probable positive colonies from the experimental plate, culture them in liquid LB overnight for mini-prep.

Mini-prep Preparation: R0010 and B0015

  • Culture R0010 and B0015 positive colonies in liquid LB overnight for mini-prep.
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