IGEM:Peking/2007/Count-Conjugation-Notebook/2007-8-6

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:

1. OriT_J23066_pSB1A2-1 @ EcoRI/SpeI
2. OriT_J23066_pSB1A2-1 @ EcoRI/SpeI
3. OriT_J23066_pSB1A2-2 @ EcoRI/SpeI
4. OriT_J23066_pSB1A2-2 @ EcoRI/SpeI
5. OriT_pSB1A2 -1 @ EcoRI/XbaI
6. OriT_pSB1A2 -1 @ EcoRI/XbaI
7. OriT
8. pSB1A2
9. marker （DL2000 plus)

electrophoresis result after gel extraction

• from left to right:

1. OriT_J23066_pSB1A2-1 @ EcoRI/SpeI (fragment)
2. OriT_pSB1A2 @ XbaI/PstI (vector)
3. 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.

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.