2009.9.3
PCR T3polymerase
| Phusion | 0.5ul
|
| Primer F | 1.25ul
|
| Primer R | 1.25ul
|
| 1ul
|
| HF Buffer | 10ul
|
| ddH2O | 32ul
|
| dNTP | 4ul
|
PCR a gradient 58, 60, 62, 65, 67
GEL Purification.
Enzyme Digestion
| PCR product | 10ul
|
| EcoRI | 1ul
|
| PstI | 1ul
|
| 10xH Buffer | 2ul
|
| ddH2O | 6ul
|
2009.9.4
Purify the product of enzyme digestion.
Ligation:
pSB1K3 backbone
T3 polymerase
Pick colonies of the T7p-CI 1-1M.
PCR assessment:
Shake each one of the correct colonies in the incubator.
2009.9.5
Miniprep 6 of the T3polymerase colonies.
5 of them are red.
Enzyme Digestion assessment of the remaining one.
At the same time PCR with the Sequencing primer for assessment.
Enzyme Digestion again to confirm:
XhoI, NotI, (XbaI, SpeI), (XbaI, SpeI, HindIII) for assessment
It is not a correct colony.
2009.9.6
PCR again the 3 counter plasmid. Same protocol.
At the same time use the PCR product of last time to do nested PCR with Standard primer.
No result for the nested PCR.
Again PCR 3 counter plasmid
Purification;
Enzyme Digestion one the PCR products with EcoRI and SpeI.
Ligation:
T3 polymerase PCR product into pEASY-BLUNT.
T3 polymerase PCR product after digestion into pSB1A2(ES).
Transformation.
Enzyme Digestion of the T7p-CI into 1-1M for Enzyme digestion assessment:
Looks like no one is correct in size.
2009.9.8
Pick 10 colonies from the pEASY-BLUNT plate for assessment.
Pick 5 colonies from the pSB1A2 plate.
Miniprep
Enzyme Digestion with EcoRI and PstI
None is correct!!!
2009.9.9
Enzyme Digestion of the 3 Counter plasmid with EcoRI and NheI, Gel purification of the correct insert that contains the T3 pol.
PCR using the purified insert as a template.
PCR 2 tubes with Phusion, one without DMSO the other with DMSO.
Gel purification of the two bands.
PCR again using the Standard primer and product of the first cycle of PCR as template.
GEL:
It seems that the size is not correct.
Purification and use some to confirm the size.
It turns out that the size is actually correct.
The product is digested with by XbaI and PstI directly.
Ligation:
T3 pol(XP) into pSB1A2
Transformation.
2009.9.11
Pick 10 colonies of the T3pol clone. No band
Mniprep 5 plasmids.
Digest with EcoRI and PstI for assessment
It seems right for NO. 1,2,3,5.
Digest with:
(ExoRI and PstI)
(EcoRI and SpeI)
(XbaI and PstI)
(XbaI and SpeI)
PCR with:
Sequencing primer
Standarlize primer
Universal Primer
For assessment
PCR
Enzyme Digestion
All phenomenon shows that this time it is correct.
Send for sequence, However, the forward primer has no signal, and the reverse primer shows more than one binding site. It is weird.
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