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

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)

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)

• 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:

2. marker

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.