Team:Peking/Notebook/QZWu

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Contents

• July, 2010

• August, 2010

• September, 2010

• October, 2010

July

July

[TOP]

7.2

1. Dissolve J23101, J23116, J23103 parts from the well and transformed them into trans 5α.

7.3

1. Anneal PmerT with Forward and reverse primers.

2. Ligase PmerT with E0840.

3. Transform.

7.4

1. Miniprep pc plasmids and PmerT-E0840.

7.5

1. Ligase RBS and merR, then transform.

2. Digest pc plasmids with EcoRI and SpeI.

7.6

1. Miniprep RBS-merR plasmid.

2. Digest the plasmid with XbaI and PstI.

7.7

1. Ligase pc with RBS-merR, then transform.

2. Confirm the product by PCR.

7.8

1. Miniprep pc-RBS-merR plasmid.

7.9

1. Confirm the product by PCR.

7.10

1. Digest Psb3K3 backbone with EcoRI and PstI.

2. Help ZTZ with his Pbad-E0840.

7.11

1. Miniprep pc-RBS-merR plasmid and digest them with EcoRI and PstI.

7.12

1. Ligase pc-RBS-merR with Psb3K3 backbone.

2. Transform.

3. For I’ve been confused about all the plasmids I can only PCR them all to test which ones are correct.

7.13-7.22

Keep on the construction of ps-RBS-merR~

A little frustrated, but things will be fine!

7.23

Get one clone, J23103-RBS-merR.

Strategy change.

Order primers that consist of pc and RBS sequences.

Use J23103 –RBS-merR as template.

Try to get other clones by PCR.

7.24

1. Purify PCR products.

2. Digest them with EcoRI and PstI.

7.25

1. Ligase pc-RBS-merR with Psb1A2 and Psb3K3 backbone.

2. Transform.

7.26

1. Miniprep plasmids.

2. Send them for sequencing.

7.27

Cannot wait for the sequencing results, start function test.

1. Prepare cells with PmerT-E0840 into competent cells.

2. Transform pc-RBS-merR plasmids with different resistance into them.

7.28

Test GFP expression level.

For the first time, there are only 5 Hg concentration gradients and no repeats.

Get data, sort of weird.

No significantly differences.

Happy anyway.

7.29

This time 10 gradients and 3 repeats.

The GFP of J23103 is extraordinarily higher than others. And no differences can be observed between the others.

7.30

Try again~

7.31

Sequencing results come. Bad news: It seems that the products were polluted by merR.

August

[TOP]

8.1

Construct parts: PmerT and PpbrA. To lift spirit.

1. Digest them and Psb1C3. 2. 3. Ligase them. 4. 5. Transform. 6.

2/8/2010

1. Incubate. 2. 3. Miniprep. 4. 5. Sequencing. 6.

8.3

Parts are right.

8.4-8.12

Home, sweet home.

Though, I’ve spent most of the time at EXPO.

8.13

Restart from the beginning.

1. PCR with my specialized primers and J23103 template.

2. Purify the products.

3. Digest.

8.16

It’s time to speed up a little.

1. Ligase with backbones Psb3K3 and Psb1A2.

2. Transform. 18 plates

8.15

1. Pick 6 candidates from each plate. Colony PCR. So many PCR tubes~

2. Miniprep those have right bands.

3. Send some for sequencing.

8.16

Digest for Psb1A2 and Psb3K3 backbones.

It’s difficult to prepare the Psb3K3 backbone. I hate low-copy plasmids.

8.17

Sequencing results come. Very, very bad news: All I got were tubes of merR and a tube of J23103.

It seems that merR pollution is still going on, and I put too much template in the PCR system.

8.18-8.21

Restart. I need to hurry.

8.22

J23108 turns out to be right, on both backbones.

8.23

J23101, J23109, J23112 have right candidates on Psb3K3 backbone.

Transform the plasmids into competent cells.

8.24

1. Pick some from plates, incubate.

2. Miniprep, CAREFULLY.

3. Digest with E, P.

8.25

1. Ligase with Psb1A2. Transform.

2. J23114, J23117 has right candidates on Psb3K3 backbone, do the same to change the backbone.

8.26

1. Miniprep and get J23101, J23109, J23112 on Psb1A2 backbone.

2. Digest J23114, J23117 with E, P.

8.27

1. J23116 has right candidates on Psb1A2 backbone. For I’ve got many right ones, I can skip the transformation, the incubation, the miniprep, and digest one tube of plasmids directly. Good.

8.28

1. Ligase and transform.

8.29

Miniprep.

8.30

Managed to get all clones I need before term begins.

1. Prepare cells with PmerT-E0840 into competent cells.

2. Transform pc-RBS-merR plasmids with different resistance into them.

8.31-9.2

Preliminary experiment.

Test GFP expression level.

There are 5 Hg concentration gradients and no repeats.