Team:PKU Beijing/Notebook/Bistable/Shan Shen

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{{PKU_Beijing/Sidebar_Notebook}}
{{PKU_Beijing/Sidebar_Notebook}}
{{PKU_Beijing/Header2}}
{{PKU_Beijing/Header2}}
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[[Team:PKU_Beijing/Notebook|Notebook]] > [[Team:PKU_Beijing/Notebook/Bistable|Bistable]] > [[Team:PKU_Beijing/Notebook/Bistable/Shan_Shen|Shan Shen's Note]]
 +
==='''2009.7.23'''===
 +
 +
'''11:30'''<br>
 +
Recycle all the inserts of SupD and terminator.
 +
 +
'''12:20'''<br>
 +
Digest high-copy bi-stable plasmids.<br>
 +
{|cellpadding=5
 +
|Total||20μL
 +
|-
 +
|Plasmids||8μL
 +
|-
 +
|EcoR1||1μL
 +
|-
 +
|Pst1||1μL
 +
|-
 +
|Buffer||2μL
 +
|-
 +
|ddH2O||8μL
 +
|}
 +
 +
'''12:30'''<br>
 +
Start to digest.
 +
 +
'''12:40'''<br>
 +
Ligation of SupD+terminator and 1-18A (strongest promoter).<br>
 +
{|cellpadding=5
 +
|Total||10μL
 +
|-
 +
|Vectors||1μL
 +
|-
 +
|Inserts||7μL
 +
|-
 +
|T4 ligase||1μL
 +
|-
 +
|Buffer||1μL
 +
|}
 +
 +
'''16:50'''<br>
 +
Electrophoresis to recycle the high-copy bi-stable digestion products.<br>
 +
The order and the amount of the samples: control plasmids 2μL, digestion products, 20μL marker 5μL.
 +
 +
==='''2009.7.24'''===
 +
 +
[[Image:PKU_20090724_Shan_Shen_1.JPG|400px]]
 +
 +
'''10:30'''<br>
 +
There are six clones on the plate. Shake those clones in the incubator.<br>
 +
PCR clones to test if they are correct.<br>
 +
{|cellpadding=5
 +
|Total||10μL
 +
|-
 +
|Template||Clones
 +
|-
 +
|dNTP||2μL
 +
|-
 +
|Buffer||1μL
 +
|-
 +
|Taq||0.5μL
 +
|-
 +
|For-primer||0.5μL
 +
|-
 +
|Rev-primer||0.5μL
 +
|-
 +
|ddH2O||5.5μL
 +
|}
 +
The results are not very good.
 +
 +
'''14:00'''<br>
 +
Transform the plasmids PKD46 from France in two tubes.
 +
 +
'''22:40'''<br>
 +
Miniprep 1-18A + SupD + terminator.
 +
 +
{|cellpadding=5
 +
|Number of the plasmids||Concentration(ng/μL)
 +
|-
 +
|Colony 1||223
 +
|-
 +
|Colony 2||355.05
 +
|-
 +
|Colony 3||148.15
 +
|-
 +
|Colony 4||124.33
 +
|-
 +
|Colony 5||113.34
 +
|-
 +
|Colony 6||146.85
 +
|}
 +
 +
==='''2009.7.28'''===
 +
 +
'''00:20'''<br>
 +
Digest 1-18A + SupD +terminator plasmids to test if they are correct. Digest them into vectors as well.<br>
 +
Digest into inserts:<br>
 +
{|cellpadding=5
 +
|Total||20μL
 +
|-
 +
|Plasmids||2μL
 +
|-
 +
|EcoR1||1μL
 +
|-
 +
|Pst1||1μL
 +
|-
 +
|Buffer||2μL
 +
|-
 +
|ddH2O||14μL
 +
|}
 +
Digest into vectors:<br>
 +
{|cellpading=5
 +
|Total||20μL
 +
|-
 +
|Plasmids||3μL
 +
|-
 +
|EcoR1||1μL
 +
|-
 +
|Pst1||1μL
 +
|-
 +
|Buffer||2μL
 +
|-
 +
|ddH2O||13μL
 +
|}
 +
PCR at the same time:<br>
 +
{|cellpadding=5
 +
|Total||10μL
 +
|-
 +
|Template||0.5μL
 +
|-
 +
|For||0.5μL
 +
|-
 +
|Rev||0.5μL
 +
|-
 +
|Mix||5μL
 +
|-
 +
|ddH2O||3.5μL
 +
|}
 +
 +
'''1:00'''<br>
 +
Start to digest.
 +
 +
'''1:30'''<br>
 +
Start to PCR.
 +
 +
'''12:00'''<br>
 +
Electrophoresis to test if the PCR products and digestion products are correct.<br>
 +
The order and the of the samples: marker, PCR products 1, PCR 2, PCR 3, PCR4, PCR5, PCR6, digestion control plasmids, digestion1, digestion2, digestion3, digestion4, digestion5, digestion 6.
 +
 +
Results:<br>
 +
[[Image:PKU_20090728_Shan_Shen_1.JPG|400px]]
 +
 +
'''16:00'''<br>
 +
Transfrom pKD3, pcp 20 to DH5a.<br>
 +
Digest 1-18C into vectors.<br>
 +
{|cellpadding=5
 +
|Total||20μL
 +
|-
 +
|Plasmids||3μL
 +
|-
 +
|Spe1||1μL
 +
|-
 +
|Pst1||1μL
 +
|-
 +
|Buffer||2μL
 +
|-
 +
|ddH2O||13μL
 +
|}
 +
 +
'''20:00'''<br>
 +
Electrophoresis the digestion products:<br>
 +
The order and the amount of the samples: marker 10μL, plasmids control 2μL, digestion 1 15μL, digestion2 15μL.<br>
 +
Results:<br>
 +
The digestion products are correct.
 +
 +
==='''2009.7.29'''===
 +
 +
'''10:00'''<br>
 +
Recycle the digestion products.<br>
 +
Miniprep the plasmids of 1-18C and PYFP.
 +
 +
'''13:30'''<br>
 +
Digest plasmids PYFP from France.<br>
 +
{|cellpadding=5
 +
|Total||20μL
 +
|-
 +
|Plasmids||4μL
 +
|-
 +
|EcoR1||1μL
 +
|-
 +
|Kpn1||1μL
 +
|-
 +
|Buffer||2μL
 +
|-
 +
|ddH2O||12μL
 +
|}
 +
 +
'''13:48'''<br>
 +
Start to digest.
 +
 +
'''15:00'''<br>
 +
Transform psp20 into DH5a.<br>
 +
Ligation of 1-18C backbone and SupD+ terminator inserts.
 +
 +
'''15:30'''<br>
 +
PCR to standardize the PYFP plasmids.<br>
 +
Take a gradient from 54 centigrade to 58 centigrade.<br>
 +
{|cellpading=5
 +
|Total||50μL
 +
|-
 +
|Template||0.2μL
 +
|-
 +
|dNTP||4μL
 +
|-
 +
|Buffer||10μL
 +
|-
 +
|Phusion||0.5μL
 +
|-
 +
|For-primer||1.25μL
 +
|-
 +
|Rev-primer||1.25μL
 +
|-
 +
|ddH2O||32.8μL
 +
|}
 +
 +
'''17:30'''<br>
 +
Digest the high-copy bi-stable PCR products.<br>
 +
{|cellpadding=5
 +
|Total||20μL
 +
|-
 +
|Fragments||5μL
 +
|-
 +
|EcoR1||1μL
 +
|-
 +
|Kpn1||1μL
 +
|-
 +
|Buffer||2μL
 +
|-
 +
|ddH2O||10μL
 +
|}
 +
 +
'''19:30'''<br>
 +
Electrophoresis the digestion products and PCR products.<br>
 +
The order of the samples: marker, plasmids control, digestion products, PCR products under 54 centigrade, PCR products under 56 centigrade, PCR products under 58 centigrade.<br>
 +
Results:<br>
 +
[[Image:PKU_20090729_Shan_Shen_1.JPG|400px]]<br>
 +
Digestion products are weird, while the PCR products under 58 centigrade are specialized. As a result, recycle the PCR products under 58 centigrade.
 +
 +
'''21:55'''<br>
 +
Digest PYFP plasmids.<br>
 +
{|cellpadding=5
 +
|Total||20μL
 +
|-
 +
|Plasmids||5μL
 +
|-
 +
|EcoR1||1μL
 +
|-
 +
|Kpn1||1μL
 +
|-
 +
|Buffer||2μL
 +
|-
 +
|ddH2O||10μL
 +
|}
==='''2009.7.30'''===
==='''2009.7.30'''===

Latest revision as of 02:45, 22 October 2009
 
Notebook > Bistable > Shan Shen's Note

2009.7.23

11:30
Recycle all the inserts of SupD and terminator.

12:20
Digest high-copy bi-stable plasmids.

Total20μL
Plasmids8μL
EcoR11μL
Pst11μL
Buffer2μL
ddH2O8μL

12:30
Start to digest.

12:40
Ligation of SupD+terminator and 1-18A (strongest promoter).

Total10μL
Vectors1μL
Inserts7μL
T4 ligase1μL
Buffer1μL

16:50
Electrophoresis to recycle the high-copy bi-stable digestion products.
The order and the amount of the samples: control plasmids 2μL, digestion products, 20μL marker 5μL.

2009.7.24

PKU 20090724 Shan Shen 1.JPG

10:30
There are six clones on the plate. Shake those clones in the incubator.
PCR clones to test if they are correct.

Total10μL
TemplateClones
dNTP2μL
Buffer1μL
Taq0.5μL
For-primer0.5μL
Rev-primer0.5μL
ddH2O5.5μL

The results are not very good.

14:00
Transform the plasmids PKD46 from France in two tubes.

22:40
Miniprep 1-18A + SupD + terminator.

Number of the plasmidsConcentration(ng/μL)
Colony 1223
Colony 2355.05
Colony 3148.15
Colony 4124.33
Colony 5113.34
Colony 6146.85

2009.7.28

00:20
Digest 1-18A + SupD +terminator plasmids to test if they are correct. Digest them into vectors as well.
Digest into inserts:

Total20μL
Plasmids2μL
EcoR11μL
Pst11μL
Buffer2μL
ddH2O14μL

Digest into vectors:

Total20μL
Plasmids3μL
EcoR11μL
Pst11μL
Buffer2μL
ddH2O13μL

PCR at the same time:

Total10μL
Template0.5μL
For0.5μL
Rev0.5μL
Mix5μL
ddH2O3.5μL

1:00
Start to digest.

1:30
Start to PCR.

12:00
Electrophoresis to test if the PCR products and digestion products are correct.
The order and the of the samples: marker, PCR products 1, PCR 2, PCR 3, PCR4, PCR5, PCR6, digestion control plasmids, digestion1, digestion2, digestion3, digestion4, digestion5, digestion 6.

Results:
PKU 20090728 Shan Shen 1.JPG

16:00
Transfrom pKD3, pcp 20 to DH5a.
Digest 1-18C into vectors.

Total20μL
Plasmids3μL
Spe11μL
Pst11μL
Buffer2μL
ddH2O13μL

20:00
Electrophoresis the digestion products:
The order and the amount of the samples: marker 10μL, plasmids control 2μL, digestion 1 15μL, digestion2 15μL.
Results:
The digestion products are correct.

2009.7.29

10:00
Recycle the digestion products.
Miniprep the plasmids of 1-18C and PYFP.

13:30
Digest plasmids PYFP from France.

Total20μL
Plasmids4μL
EcoR11μL
Kpn11μL
Buffer2μL
ddH2O12μL

13:48
Start to digest.

15:00
Transform psp20 into DH5a.
Ligation of 1-18C backbone and SupD+ terminator inserts.

15:30
PCR to standardize the PYFP plasmids.
Take a gradient from 54 centigrade to 58 centigrade.

Total50μL
Template0.2μL
dNTP4μL
Buffer10μL
Phusion0.5μL
For-primer1.25μL
Rev-primer1.25μL
ddH2O32.8μL

17:30
Digest the high-copy bi-stable PCR products.

Total20μL
Fragments5μL
EcoR11μL
Kpn11μL
Buffer2μL
ddH2O10μL

19:30
Electrophoresis the digestion products and PCR products.
The order of the samples: marker, plasmids control, digestion products, PCR products under 54 centigrade, PCR products under 56 centigrade, PCR products under 58 centigrade.
Results:
PKU 20090729 Shan Shen 1.JPG
Digestion products are weird, while the PCR products under 58 centigrade are specialized. As a result, recycle the PCR products under 58 centigrade.

21:55
Digest PYFP plasmids.

Total20μL
Plasmids5μL
EcoR11μL
Kpn11μL
Buffer2μL
ddH2O10μL

2009.7.30

9:00
Electrophoresis to test the products of the digestion.
The order of the samples: marker, plasmids control, digestion products.
Results:
PKU 20090730 Shan Shen 1.JPG
It is difficult to tell the digestion fragments, maybe because of the poor concentration of the plasmids.

20:00
Digest the PYFP plasmids into vectors.
Digest to standardize:

Total20μL
Plasmids7μL
Spe11μL
Pst11μL
Buffer2μL
ddH2O9μL

Digest to connect with high-copy bi-stable fragments.

Total20μL
Plasmids10μL
EcoR11μL
Kpn11μL
Buffer2μL
ddH2O6μL

Digest the standardizing PCR fragments into inserts:

Total20μL
Plasmids17μL
Xba11μL
Buffer2μL

Digest T7P + 1-12M to get the high-copy backbone.

Total20μL
Plasmids4μL
Xba11μL
Spe11μL
Buffer2μL
ddH2O12μL

2009.7.31

10:30
Electrophoresis the digestion products.
The order of the samples: marker, digestion products for backbone, single digestion products of the standardized fragments, double digestion products of PYFP cut by Kpn1 and EcoR1, double digestion products of PYFP cut by Spe1 and Pst1.
Results:
PKU 20090731 Shan Shen 1.JPG
Recycle the fragments.

12:00
Digest the single digestion products of PYFP by another enzyme.

Total30μL
Fragments25.5μL
Apal11.5μL
Buffer3μL

12:40
Start to digest.

14:00
Miniprep.

Number of the plasmidsConcentration(ng/μL)
PKD4653.68
High-copy bi-stable102.61
PKD3139.91
Psp2072.955
PYFP100.805

16:00
Digest high-copy bi-stable plasmids.

Total20μL
Plasmids15μL
Xba11μL
Nhe11μL
Spe11μL
Buffer2μL

16:30
Start to digest.

16:30
Start to recycle the fragments digested by ApaL1

17:30
Link the standard fragments with PYFP vectors, also high-copy bi-stable fragments with PYFP vectors.

Total10μL
Vectors2μL
Inserts6μL
T4 ligase1μL
Buffer1μL

22:00
Electrophoresis to test the digestion products.
The order of the samples: marker, high-copy control plasmids, digestion products.
Results:
Digestion is successful. Recycle the fragments of about 3kb.

22:30
Transform the ligation products into DH5a.

2009.8.1

00:30
Start to incubate.

13:30
There is no colony on the plates.

21:00
Digest the high-copy bi-stable plasmids and low-copy bi-table plasmids.
High-copy bi-stable plasmids:

Total20μL
Plasmids2μL
EcoR11μL
Spe11μL
Buffer2μL
ddH2O13μL

Low-copy bi-stable plasmids:

Total20μL
Plasmids7μL
EcoR11μL
Nhe11μL
Spe11μL
Buffer2μL
ddH2O8μL

PYFP:

Total20μL
Plasmids10μL
EcoR11μL
Kpn11μL
Buffer2μL
ddH2O6μL
Total20μL
Plasmids10μL
Pst11μL
Spe11μL
Buffer2μL
ddH2O6μL

Digest T7P + 1-12M for backbone:

Total20μL
Plasmids4μL
EcoR11μL
Spe11μL
Buffer2μL
ddH2O12μL

22:00
Start to digest.

2009.8.2

1:00
Electrophoresis to test the digestion products.

The order of the samples: marker, high-copy plasmids, high-copy digestion products, low-copy plasmids, low-copy digestion products, PYFP plasmids, PYFP digestion products by EK, PYFP digestion products by SP, T7P+1-12M plasmids, T7P+1-12M digestion products.
Results:
PKU 20090802 Shan Shen 1.JPG
Electrophoresis the fragments used in the ligation the day before.
Results:
PKU 20090802 Shan Shen 2.JPG
The concentrations of the fragments are too low, so that cause the ligation to fail.

2:00
Recycle the fragments.

4:00
Connect the bi-stable fragments, standard fragments with PYFP vectors.
Connect the high-copy bi-stable fragments and low-copy bi-stable fragments with the high-copy backbone.

Total10μL
Vectors2μL
Inserts6μL
T4 ligase1μL
Buffer1μL

13:00
Transform those ligation products into DH5a.

2009.8.3

8:30
There are many colonies on all the plates.

9:30
Start to shake the colonies in the incubator.

11:30
PCR to test the colonies at the same time.

Total20μL
TemplateColonies
For1μL
Rev1μL
Mix10μL
ddH2O8μL

17:00
Electrophoresis to test the PCR products.
The order of the samples: standardized PYFP, marker, PYFP with bi-stable parts, high-copy bi-stable parts, marker, low-copy bi-stable parts.

23:00
Miniprep.

Number of the plasmidsConcentration(ng/μL)
PYFP with bi-stable parts 1123.9
PYFP with bi-stable parts 292.54
PYFP with bi-stable parts 3109.42
PYFP with bi-stable parts 544.68
Low-copy bi-stable parts 381.195
Low-copy bi-stable parts 4321.055
High-copy bi-stable parts 3160.98
High-copy bi-stable parts 4100.805
PYFP with standardized fragments 555.82

2009.8.4

2:30
Digest and PCR those plasmids to test if they are correct.
PYFP with bi-stable parts:

Total20μL
Plasmids3μL
EcoR11μL
Kpn11μL
Buffer2μL
ddH2O13μL

High-copy and low-copy bi-stable parts with high-copy backbone:

Total20μL
Plasmids3μL
EcoR11μL
Pst11μL
Buffer2μL
ddH2O13μL

PYFP vectors with standardized fragments:

Total20μL
Plasmids6μL
Pst11μL
Spe11μL
Buffer2μL
ddH2O1μL

This is to test if the restriction enzyme sites are successfully been erased.
PCR to test standardization:

Total20μL
Template1μL
For1μL
Rev1μL
Mix10μL
ddH2O7μL

10:30
Electrophoresis to test if the PCR and digestion products are correct.
The order of the samples:
Marker, PYFP with bi-stable parts plasmids control, PYFP with bi-stable digestion products 1,2,3,5, PCR products of standardization, the digestion products of standardization.
Marker, low-copy bi-stable parts, high-copy bi-stable parts.
Results:
PKU 20090804 Shan Shen 1.JPG
The PFYP with the bi-stable parts are correct.

16:00
Make up SOB.
100ML

2009.8.5

10:00
Prepare competent cells for electrical transformation.

20:00
Digest the PYFP with bi-stable parts to get the inserts.

Total50μL
Plasmids10μL
Sap11.5μL
Sph11.5μL
Buffer5μL
ddH2O32μL

2009.8.6

12:30
Recycle the digestion products.

13:30
Prepare SOC.

20:00
Electroporation:
Parameters: 50μLwith 400ng DNA fragments.
2.5kV, 25mF, 200ohms.
Incubate in SOC for 2h.

2009.8.7

There is one colony on the plate, but it is the result of reconnection of the plasmids.

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