IGEM:Peking/2007/Count-Conjugation-Notebook/2007-8-15
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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:
- DL 2000 plus marker
- R0010.J01008 @ SpeI/PstI purified digestion product
- 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:
- B0015-1 @ XbaI/PstI purified digestion product
- B0015-2 @ XbaI/PstI purified digestion product
- E0040.B0015 @ EcoR/XbaI purified digestion product
- 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.