IGEM:Peking/2007/Count-Conjugation-Notebook/2007-8-6
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Tandem OriT by Qu Mingzhi
mini-prep OriT_J23066_pSB1A2
- using Transgen mini plasmid puriflication kit.
- 30µL after purflication
mini-prep double digesting test result & gel extraction purification
- Digesting OriT_J23066_pSB1A2 with EcoRI/SpeI. (as Standard Assembly fragment)
- Digesting OriT_pSB1A2 with EcoRI/XbaI. (as Standard Assembly vector)
- OriT_J23066_pSB1A2 Digestion system contains:
4 µl 10*H 1 µl EcoRI 1 µl SpeI 20 µl Plasmid 14 µl dH20 -------------------------- 40 µl Total
- OriT_pSB1A2 Digestion system contains:
4 µl 10*M 1 µl EcoRI 1 µl XbaI 20 µl Plasmid 14 µl dH20 -------------------------- 40 µl Total
electrophoresis result before gel extraction
- from left to right:
- OriT_J23066_pSB1A2-1 @ EcoRI/SpeI
- OriT_J23066_pSB1A2-1 @ EcoRI/SpeI
- OriT_J23066_pSB1A2-2 @ EcoRI/SpeI
- OriT_J23066_pSB1A2-2 @ EcoRI/SpeI
- OriT_pSB1A2 -1 @ EcoRI/XbaI
- OriT_pSB1A2 -1 @ EcoRI/XbaI
- OriT
- pSB1A2
- marker (DL2000 plus)
electrophoresis result after gel extraction
- from left to right:
- OriT_J23066_pSB1A2-1 @ EcoRI/SpeI (fragment)
- OriT_pSB1A2 @ XbaI/PstI (vector)
- marker (DL2000 plus)
Ligation: OriT_J23066_pSB1A2 -> OriT_pSB1A2
- Ligate the OriT_J23066_pSB1A2 fragment and OriT_pSB1A2 vector
- test new super fast T4 DNA ligase
- super fast T4 DNA ligase Ligation system contains:
2 µl F-OriT_pSB1A2 vector 2 µl J23066 fragment 0.5 µl ''super fast T4 DNA ligase'' 1 µl 10X T4 ligase buffer 4.5 µl dH20 -------------------------- 10 µl Total
- 16℃ for 10 min!
- Ordinary ligation system contains:
2 µl F-OriT_pSB1A2 vector 2 µl J23066 fragment 1 µl ''super fast T4 DNA ligase'' 1 µl 10X T4 ligase buffer 4 µl dH20 -------------------------- 10 µl Total
- 16℃ over night
transformation OriT_J23066_OriT_pSB1A2
- transformation OriT_J23066_OriT_pSB1A2 ligate by super fast T4 DNA ligase.
- Culture at Amp+ plate for 12 hours.
- NEXT DAY: 100+ cloney received.
Lock & Key By Yu Tao
oriT Knock Out
- By Xu Anting
Re-do pSC101 PCR
- Use purified plasmid pSC101 as Template
- Temperature gradient : 47-61℃
- Distribute as 10µl per tube * 8 temperature * 2 enzymes
70 µl ddH2O 8 µl 2.5 mM dNTP 0.5 µl pSC101 Template 5 µl Primer_psC101(1, 2)_Sense 5 µl Primer_psC101(1, 2)_Antisense 10 µl 10* Taq/Pfu buffer 1 µl Taq/Pfu -------------------------- 100 µl Total
- Result: Both of up- and downstream primers only show bands at 47℃ using Pfu.
pKO3 vector preparation
- After plasmid purification, using BamHI and BamHI/SalI to digest the plasmid for 5 hours. Then separate and purify them in agarose gel.