IGEM:Peking/2007/Count-Conjugation-Notebook/2007-8-15

Tandem OriT by Qu Mingzhi

transformation R-OriT & S-OriT

• Use pEASY-3 as cloning vector .(Amp+)
• tramsformation system contains :3uL R-OriT, S-OriT PCR product(after purflication), 1uL pEasy-T3
• the Culture Dish(LB/Amp+) are trated with 5uL 1M IPTG & 40uL 40mg/mL x-Gal.
• NEXT DAY: 10+ white cloney received.

Amplification Culture of E0240

• select Positive E0240 Colonies from Plate,Culture in liquid LB,waiting for mini-prep.

Lock & Key By Yu Tao

R0010.J01008 and R0040.J01010 Digestion Product Purification

• Use Transgen EasyPure PCR Purification Kit for R0010<-J01008 and Quick Gel Extraction Kit for R0040<--J01010.
• 30uL per tube after purflication, one tube, respectively.

Electrophorsis Result

• from left to right:

1. DL 2000 plus marker
2. R0010.J01008 @ SpeI/PstI purified digestion product
3. R0040.J01010 @ EcoRI/SpeI purified digestion product

Ligation: R0010.J01008<-B0015 and R0040.J01010->E0040.B0015

Recheck previous B0015 fragment and E0040.B0015 vector result

• from left to right:

1. B0015-1 @ XbaI/PstI purified digestion product
2. B0015-2 @ XbaI/PstI purified digestion product
3. E0040.B0015 @ EcoR/XbaI purified digestion product
4. DL2000 plus marker

• Conclusion: Use the B0015-2 @ XbaI/PstI purified digestion product and E0040.B0015 @ EcoR/XbaI purified digestion product.

• Ligate the R0040.J01010 fragment and E0040.B0015 vector, as well as the B0015 fragment and R0010<-J01008 vector.
• Ligation system contains:

7 µl       R0040.J01010 fragment   /   3 µl       B0015 fragment
1 µl       E0040.B0015 vector      /   1 µl       R0010<-J01008 vector  
0.5 µl     Super T4-Ligase
1 µl       10 X ligation buffer
0 µl       ddH20                   /   4 µl       ddH20
--------------------------
9.5 µl     Total

• The negative control group contains no fragment but ddH2O instead.
• 10min at 16℃.

Transformation: R0010.J01008<-B0015 and R0040.J01010->E0040.B0015

• Transform all ligation products into 100 µl DH5α competent cells.
• Culture all R0010.J01008<-B0015 cells at Amp+ LB plate and all R0040.J01010->E0040.B0015 cells at Kan+ LB plate for 12 hours.
• Result to be seen tomorrow.

New competent cells preparation

• Prepare Competent Cells III

Competent Cells III efficiency test

• Trasform 1uL R0010 plasmid into competent cells for test.

                            | Competent Cells II | Competent Cells III | Competent Cells III
------------------------------------------------------------------------------------------------
           R0010            |        1 uL        |        1 uL         |       0 uL
------------------------------------------------------------------------------------------------
           ddH2O            |        0 uL        |        0 uL         |       1 uL
------------------------------------------------------------------------------------------------
antibiotics resistency test |        Amp         |        Amp          |  Amp/Kan,respectively

Double Digestion: R0040.J01010

• Because the purified digestion product of R0040.J01010 is almost unseen in the electrophoresis result, I decide to redo it once more.
• Digesting R0040.J01010 with EcoRI/SpeI.
• Each digestion system contains:

4 µl       10*H
1 µl       EcoRI
1 µl       SpeI
25 µl      Plasmid
9 µl       ddH20
--------------------------
40 µl      Total

• 37℃ culutre overnight.