Team:PKU Beijing/Notebook/AND Gate 2/Min Lin

From 2009.igem.org

(Difference between revisions)
(→2009.9.3)
Line 149: Line 149:
All phenomenon shows that this time it is correct.<br>
All phenomenon shows that this time it is correct.<br>
Send for sequence, However, the forward primer has no signal, and the reverse primer shows more than one binding site. It is weird.
Send for sequence, However, the forward primer has no signal, and the reverse primer shows more than one binding site. It is weird.
 +
 +
==='''2009.9.15'''===
 +
 +
Transformation from the part distribution the P2 activator PhiR73 delta and one of its promoter PO.
 +
 +
Plate overnight.
 +
 +
==='''2009.9.16'''===
 +
 +
Pick one of the PhiR73 delta colony and PO promoter colony, shake in the incubator.
 +
 +
Miniprep PhiR73 delta and PO promoter plasmid.
 +
 +
Use Phusion to PCR the PhiR73 delta plasmid over night.
 +
 +
{|cellpadding=5
 +
|Phusion||0.5ul
 +
|-
 +
|PointMutation For||1.25ul
 +
|-
 +
|PointMutation Rev1||1.25ul
 +
|-
 +
|Template||1ul
 +
|-
 +
|HF Buffer||10ul
 +
|-
 +
|ddH2O||32ul
 +
|-
 +
|dNTP||4ul
 +
|}
 +
 +
 +
{|cellpadding=5
 +
|Phusion||0.5ul
 +
|-
 +
|PointMutation For||1.25ul
 +
|-
 +
|PointMutation Rev2||1.25ul
 +
|-
 +
|Template||1ul
 +
|-
 +
|HF Buffer||10ul
 +
|-
 +
|ddH2O||32ul
 +
|-
 +
|dNTP||4ul
 +
|}
 +
 +
The first PCR introduces one point mutation and the second introduces two.
 +
 +
Enzyme Digestion of the PO promoter plasmid:
 +
SpeI 1ul
 +
PstI 1ul
 +
Buffer 3 2ul
 +
Plasmid 3ul
 +
ddH2O 13ul
 +
 +
Enzyme Digestion of the E0840
 +
XbaI 1ul
 +
PstI 1ul
 +
Plasmid 5ul
 +
ddH2O 11ul
 +
Buffer 2ul
 +
 +
 +
==='''2009.9.17'''===
 +
 +
GEL of the PCR product
 +
 +
Purification of the GEL.
 +
 +
Blunting kination reaction 70 centigrade for 10min then 37 centigrade for 10min
 +
 +
Ligation.
 +
 +
GEL assessment of the PO promoter plasmid(SP digest)
 +
 +
CIAP it for 20 min.
 +
 +
Directly purify it.
 +
 +
GEL: Enzyme Digestion of E0840, gel purification of the insert.
 +
 +
Ligation:
 +
PO promoter vector 1ul
 +
E0840 insert 7ul
 +
Ligase 1ul
 +
Ligation buffer 1ul
 +
 +
Transformation:
 +
PointMutation1, PointMutation2, PO-GFP.
 +
 +
Plate
 +
 +
==='''2009.9.18'''===
 +
 +
Pick single colony of the PO-GFP and PCR to assess whether it is correct.
 +
All 5 of the colonies are correct.
 +
 +
Pick from PointMutation1, PointMutation2 plate each 5 colonies. Shake in the incubator.
 +
Pick one colony from the PO-GFP construct and shake in the incubator.
 +
 +
Miniprep the 11 tubes.
 +
 +
Enzyme Digestion:
 +
Digest PointMutation plasmids with EcoRI and PstI for assessment.
 +
Disgest PO-GFP with XbaI and PstI
 +
Get the Salicylate inducible promoter from zgs and digest with speI and pstI.
 +
 +
==='''2009.9.19'''===
 +
 +
GEL:
 +
PO-GFP and get the XP insert.
 +
 +
Sal promoter and pointmutation colonies for assessment.
 +
 +
Send pointmutation plasmid for sequencing.
 +
 +
CIAP the sal promoter vector for 20 min
 +
 +
Ligation:
 +
PO-GFP XP insert and sal promoter vector
 +
 +
Transformation.
 +
 +
 +
 +
==='''2009.9.20'''===
 +
 +
Pick colonies of the sal-PO-GFP
 +
 +
Colony PCR to assess. (get the correct colony, shake in the incubator)
 +
 +
Miniprep.
 +
 +
==='''2009.9.21'''===
 +
 +
Enzyme Digestion PM9 with XbaI and PstI.
 +
 +
GEL. Purification.
 +
 +
Ligation:
 +
Sal promoter(sp) vector
 +
PM9(XP)
 +
 +
 +
 +
Transformation
 +
 +
==='''2009.9.22'''===
 +
 +
Pick colonies for PCR assessment. Get the correct colony.
 +
 +
Get Ara-SupD from GRC.
 +
 +
Enzyme Digestion with EcoRI SpeI.
 +
 +
Shake the Sal-PM9 colony in the incubator.
 +
Shake the pSB4K5 1-7G strain in the incubator.
 +
 +
Gel: get Ara-SupD (ES insert)
 +
 +
==='''2009.9.23'''===
 +
 +
Miniprep the two plasmids.
 +
Enzyme Digestion pSal-PM9 with XbaI and PstI
 +
Digest pSB4K5 with EcoRI and PstI
 +
Gel: get pSal-PM9 (XP insert), pSB4K5 EP backbone.
 +
 +
Ligation:
 +
pSal-PM9(XP insert) 3.5ul
 +
ara-supD(ES insert )3.5ul
 +
pSB4K5 EP vector 1ul
 +
ligase 1ul
 +
ligation buffer 1ul.
 +
 +
Ligation over night.
 +
 +
==='''2009.9.24'''===
 +
 +
Transformation.
 +
 +
==='''2009.9.25'''===
 +
 +
 +
Pick colonies PCR assessment. No result. Maybe due to low copy plasmid.
 +
 +
Shake in the incubator.
 +
 +
==='''2009.9.26'''===
 +
 +
Miniprep.
 +
 +
Enzyme Digestion of the plasmids.
 +
 +
Gel assessment.
 +
 +
Get the correct colony.
 +
 +
 +
==='''2009.9.27'''===
 +
 +
Second plasmid transformation:
 +
Shake the PO-GFP strain in the incubator to OD 0.4
 +
 +
Suspend with CaCl2 and make competent cells.
 +
 +
Transformation with the correct ara-supD-pSal-PM9 plasmid.
 +
 +
Plate over night.
 +
 +
==='''2009.9.28'''===
 +
 +
Shake the transformation from last night.
 +
Shake the ORgate-GFP strain.
 +
 +
Induction.
 +
{{PKU_Beijing/Foot}}
{{PKU_Beijing/Foot}}
__NOTOC__
__NOTOC__

Revision as of 05:46, 16 October 2009
 

2009.9.3

PCR T3polymerase

Phusion0.5ul
Primer F1.25ul
Primer R1.25ul
Template1ul
HF Buffer10ul
ddH2O32ul
dNTP4ul

PCR a gradient 58, 60, 62, 65, 67

PKU 20090903 Min Lin 1.JPG

GEL Purification.

Enzyme Digestion

PCR product10ul
EcoRI1ul
PstI1ul
10xH Buffer2ul
ddH2O6ul

2009.9.4

Purify the product of enzyme digestion.

Ligation:
pSB1K3 backbone
T3 polymerase

Pick colonies of the T7p-CI  1-1M.
PCR assessment:
PKU 20090904 Min Lin 1.JPG
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.
PKU 20090905 Min Lin 1.JPG

Enzyme Digestion again to confirm:
XhoI, NotI, (XbaI, SpeI), (XbaI, SpeI, HindIII) for assessment
PKU 20090905 Min Lin 2.JPG
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.
PKU 20090906 Min Lin 1.JPG
No result for the nested PCR.

Again PCR 3 counter plasmid
PKU 20090906 Min Lin 2.JPG
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:
PKU 20090906 Min Lin 3.JPG
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
PKU 20090908 Min Lin 1.JPG
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.
PKU 20090909 Min Lin 1.JPG

Gel purification of the two bands.

PCR again using the Standard primer and product of the first cycle of PCR as template. GEL:
PKU 20090909 Min Lin 2.JPG
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.
PKU 20090909 Min Lin 3.JPG

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
PKU 20090911 Min Lin 1.JPG
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
PKU 20090911 Min Lin 2.JPG
Enzyme Digestion
PKU 20090911 Min Lin 3.JPG

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.

2009.9.15

Transformation from the part distribution the P2 activator PhiR73 delta and one of its promoter PO.

Plate overnight.

2009.9.16

Pick one of the PhiR73 delta colony and PO promoter colony, shake in the incubator.

Miniprep PhiR73 delta and PO promoter plasmid.

Use Phusion to PCR the PhiR73 delta plasmid over night.

Phusion0.5ul
PointMutation For1.25ul
PointMutation Rev11.25ul
Template1ul
HF Buffer10ul
ddH2O32ul
dNTP4ul


Phusion0.5ul
PointMutation For1.25ul
PointMutation Rev21.25ul
Template1ul
HF Buffer10ul
ddH2O32ul
dNTP4ul

The first PCR introduces one point mutation and the second introduces two.

Enzyme Digestion of the PO promoter plasmid: SpeI 1ul PstI 1ul Buffer 3 2ul Plasmid 3ul ddH2O 13ul

Enzyme Digestion of the E0840 XbaI 1ul PstI 1ul Plasmid 5ul ddH2O 11ul Buffer 2ul


2009.9.17

GEL of the PCR product

Purification of the GEL.

Blunting kination reaction 70 centigrade for 10min then 37 centigrade for 10min

Ligation.

GEL assessment of the PO promoter plasmid(SP digest)

CIAP it for 20 min.

Directly purify it.

GEL: Enzyme Digestion of E0840, gel purification of the insert.

Ligation: PO promoter vector 1ul E0840 insert 7ul Ligase 1ul Ligation buffer 1ul

Transformation: PointMutation1, PointMutation2, PO-GFP.

Plate

2009.9.18

Pick single colony of the PO-GFP and PCR to assess whether it is correct. All 5 of the colonies are correct.

Pick from PointMutation1, PointMutation2 plate each 5 colonies. Shake in the incubator. Pick one colony from the PO-GFP construct and shake in the incubator.

Miniprep the 11 tubes.

Enzyme Digestion: Digest PointMutation plasmids with EcoRI and PstI for assessment. Disgest PO-GFP with XbaI and PstI Get the Salicylate inducible promoter from zgs and digest with speI and pstI.

2009.9.19

GEL: PO-GFP and get the XP insert.

Sal promoter and pointmutation colonies for assessment.

Send pointmutation plasmid for sequencing.

CIAP the sal promoter vector for 20 min

Ligation: PO-GFP XP insert and sal promoter vector

Transformation.


2009.9.20

Pick colonies of the sal-PO-GFP

Colony PCR to assess. (get the correct colony, shake in the incubator)

Miniprep.

2009.9.21

Enzyme Digestion PM9 with XbaI and PstI.

GEL. Purification.

Ligation: Sal promoter(sp) vector PM9(XP)


Transformation

2009.9.22

Pick colonies for PCR assessment. Get the correct colony.

Get Ara-SupD from GRC.

Enzyme Digestion with EcoRI SpeI.

Shake the Sal-PM9 colony in the incubator. Shake the pSB4K5 1-7G strain in the incubator.

Gel: get Ara-SupD (ES insert)

2009.9.23

Miniprep the two plasmids. Enzyme Digestion pSal-PM9 with XbaI and PstI Digest pSB4K5 with EcoRI and PstI Gel: get pSal-PM9 (XP insert), pSB4K5 EP backbone.

Ligation: pSal-PM9(XP insert) 3.5ul ara-supD(ES insert )3.5ul pSB4K5 EP vector 1ul ligase 1ul ligation buffer 1ul.

Ligation over night.

2009.9.24

Transformation.

2009.9.25

Pick colonies PCR assessment. No result. Maybe due to low copy plasmid.

Shake in the incubator.

2009.9.26

Miniprep.

Enzyme Digestion of the plasmids.

Gel assessment.

Get the correct colony.


2009.9.27

Second plasmid transformation: Shake the PO-GFP strain in the incubator to OD 0.4

Suspend with CaCl2 and make competent cells.

Transformation with the correct ara-supD-pSal-PM9 plasmid.

Plate over night.

2009.9.28

Shake the transformation from last night. Shake the ORgate-GFP strain.

Induction.



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