From 2007.igem.org

07-07-01

1. Design the primer of PCR amplification of riboflavin operon, The restriction site of up- and down-primer are SmaI(Cfr9I) and KpnI, respectively.

2. Waiting for primer synthesis

07-07-12

1. Amplify the riboflavin opron rib(CDS) fragment

2. Test the PCR product with gel electrophoresis, the length of riboflavin operon fragment is 4kb

3. Purify the PCR product

4. Transform plasmid puc18 ADHI 1μl into E.coli Top10

5. Cultivate the transformant on LB solid media with 100μg/ml Amp for 14h at 37℃

07-07-13

1. Take 4 well-grown colonies from the media and transfer them onto LB liquid media with 100μg/ml Amp for ampliative cultivation for 10h at 37℃

2. Distill the plasmid with CTAB method

3. Test the distilled plasmid with enzymatic digest, There are totally three enzyme systems:

(1)BamHI single enzyme

(2)KpnI, SacI double enzymes

(3)XbalI, BamHI double enzymes

07-07-14

1. Test the result of enzymatic digest with gel electrophoresis

2. The result is consistent with our expectation. Keep the plasmid in 80% glycerol in -20℃

3. Digest pub18-ADHI and rib(CDS) with cfr9I

07-07-15

1. Test the digeststion product with gel electrophoresis, the length is consistent with our exception: the length of plasmid puc18-ADHI is 3.8kb and that of rib(CDS) is 4kb

2. Ligation. the carrier(puc18-ADHI) and extrogenous fragment(rib(CDS)) is mixed by 1:1, 1:2, 1:4, 1:6, respectively, the volume of ligation system is 20μl. Ligation lasts for 2h at 21℃

3. Transfer the ligation product into E.coli Top10

4. Cultivate the transformant on LB solid media with 100μg/ml Amp overnight at 37℃. At the mean time, cultivate the colon with enzymatic digest product of carrier puc18-ADHI as a blank experiment.

07-07-16

1. Observe the result of cultivation: the bacterial density is larger on the plate with ligation production on it than that with enzymatic digest product of carrier puc18-ADHI on it

2. Transfer 6 well-grown colonies onto LB liquid media with 100μg/ml Amp for ampliative cultivation for 10h at 37℃

3. Extract the plasmid of cultivated strain

4. Digest the plasmid with KpnI single enzyme

5. Test the result of enzymatic degestation with gel electrophoresis

6. The plasmid is the product of self-ligation. Ligation failed

07-07-07~19

The second time of ligation. Failed again. We will begin to do the experiment of mechanism II tomorrow.

07-07-20

1. Amplify the plasmid YIPlac211

2. Transform plasmid YIPlac211 1μl into E.coli Top10

3. Cultivate the transformant on LB solid media with 100μg/ml Amp for 24h at 37℃

07-07-21

1. Take 6 well-grown colonies from the media and transfer them into LB liquid media with 100μg/ml Amp for ampliative cultivation for 16h at 37℃

2. Distill the plasmid with CTAB method

3. Test the distilled plasmid with enzymatic digest. There are two enzyme systems:

(1)BamHI singal enzyme

(2)BamHI, XbaI double enzymes

4. Test the result of enzymatic digest with gel electrophoresis

5. The result is consistent with our expectation. Keep the plasmid in 80% glycerol in -20℃

6. Knock out the EcoRI site of plasmid YIPlac211 and the new plasmid is named YIPlac211-E

(1)digest YIPlac211 with EcoRI overnight

07-07-22

Knock out the EcoRI site of plasmid YIPlac211 and the new plasmid is named YIPlac211-E

(2)test the result of digest with gel electrophoresis

(3)The result is consistent with our expection. The plasmid is cutted by EcoRI completely

(4) Repair the 3’ cohesive end with Klenow enzyme so as to change it into a blunt end.

(5) Link the blunt-ended DNA with T4 ligase

(6) Transform the ligation product into E.coli Top10

(7) Cultivate the transferred strain on LB solid media with 100μg/ml Amp overnight at 37℃.

07-07-23

Knock out the EcoRI site of plasmid YIPlac211 and the new plasmid is named YIPlac211-E

(8) Cultivate the transformant in LB liquid media with 100μg/ml Amp for ampliative cultivation for 10h at 37℃

(9) Digest the plasmid with ScaI and BamHI, respectively.

(10) Test the result of digest with gel electrophoresis. The length of plasmid is 3.8kb, which is consistent with our expection.

07-07-24

Knock out the EcoRI site of plasmid YIPlac211 and the new plasmid is named YIPlac211-E

(11) Digest the plasmid with two enzyme systems:

 (I) NcoI and EcoRI double enzymes

 (II) NcoI single enzyme

(12) Test the result of enzymatic digest with gel electrophoresis. The result of the two enzyme systems are the same. It means the EcoRI site is knocked out successfully.

07-07-25

1. Digest the plasmid YIPlac211-E and ADHI promoter with XbalI and BamHI.

2. Ligation. The carrier(YIPlac211-E) and ADHI is mixed by 1:1, 1:3, 1:6, respectively, the volume of ligation system is 20μl. Ligation lasts for 3h at 21℃

3. Transform the ligation product into E.coli Top10

4. Cultivate the transformant on LB solid media with 100μg/ml Amp overnight at 37℃

07-07-26

Digest the carrier plasmid YIPlac211-E and plasmid which contains extrogenous fragment ADHIP with XbaI and BamHI overnight at 37℃

07-07-27

1. Do gel electrophoresis with the product of enzymatic digest and recycle the carrier and extrogenous fragment by Gel Extraction Purification

2. Purify the recycled product

(1) mix the product with 1/10 fold NaAc and 2/3 fold pre-colded absolute ethanol and keep it in -20oC for 30 min

(2) centrifuge at 13000rpm. Abandon the supernatant

(3) wash it with 100ul absolute ethanol and centrifuge at 13000rpm, abandon the supernatant. Dry it at 37oC for 15min with it cover uncovered

(4) dissolve it with 10ulTE and conserve it at -20oC

3. Ligation: the proportions of carrier and extrogenous fragment are: 1:1, 1:3, 1:6. the volume of ligation system is 20μl, 21℃, 3h. The ligation system is showed below:

1（ul) 2（ul) 3（ul）

YIPlac211-E 1 1 1

ADHI-E 1 3 6

T4 Buffer 2 2 2

ddH2O 16 14 11

T4ligase 0.4 0.4 0.4

4. Transform the products of ligation into E.coli TOP10, respectively.

5. Cultivate the transferred strain on LB solid media with 100μg/ml Amp for 16h at 37℃.

07-07-28

1. Observe the result of cultivation. There are one transformant on 1:3 and 1:6 plate, respectively

2. Culture these two transformants in LB liquid media with 100μg/ml Amp for ampliative cultivation for 12h at 37℃

3. Distill the plasmid

4. Test the distilled plasmid with enzymatic digest. there are two enzyme systems:

(1)HindIII singal enzyme

(2)BamHI, XbaI double enzymes

5. Test the result of enzymatic digest with gel electrophoresis. The result isn’t the one we want. Ligation failed

A second time of ligation.

1. Digest the plasmid YIPlac211-E and ADHI promoter with XbalI and BamHI. The digest system is showed below:

YIPlac211-E:

DNA 3ul

Buffer BamHI 4ul

XbaI 0.4ul

BamHI 1ul

ddH2O 31.5ul

total 40 ul

ASHI:

DNA 5ul

Buffer BamHI 6ul

XbaI 0.5ul

BamHI 1ul

ddH2O 47.5ul

total 60 ul

2. Ligation. The carrier(YIPlac211-E) and ADHI is mixed by 1:1.5, 1:3, 1:5.5, respectively, the volume of ligation system is 20μl. Ligation lasts for 3h at 21℃

3. Transform the ligation product into E.coli Top10

4. Cultivate the transformant on LB solid media with 100μg/ml Amp overnight at 37℃

07-07-29

Digest the carrier plasmid YIPlac211-E and plasmid which contains extrogenous fragment ADHIP with XbaI and BamHI overnight at 37℃

07-07-30

1. Do gel electrophoresis with the product of enzymatic digest and recycle the carrier and extrogenous fragment by Gel Extraction Purification

2. Purify the recycled product

(1) mix the product with 1/10 fold NaAc and 2/3 fold pre-colded absolute ethanol and keep it in -20oC for 30 min

(2) centrifuge at 13000rpm. Abandon the supernatant

(3) wash it with 100ul absolute ethanol and centrifuge at 13000rpm, abandon the supernatant. Dry it at 37oC for 15min with it cover uncovered

(4) dissolve it with 10ulTE and conserve it at -20oC

3. Ligation: the proportions of carrier and extrogenous fragment are: 1:1, 1:3, 1:6. the volume of ligation system is 20μl, 21℃, 3h. The ligation system is showed below:

1（ul） 2（ul） 3（ul）

YIPlac211-E 1 1 1

ADHI-E 1 3 6

T4 Buffer 2 2 2

ddH2O 16 14 11

T4ligase 0.4 0.4 0.4

4. Transform the products of ligation into E.coli TOP10, respectively.

5. Cultivate the transferred strain on LB solid media with 100μg/ml Amp for 16h at 37℃.

07-07-31

1. Observe the result of cultivation. There are one transformant on 1:3 and 1:6 plate, respectively

2. Culture these two transformants in LB liquid media with 100μg/ml Amp for ampliative cultivation for 12h at 37℃

3. Distill the plasmid

4. Test the distilled plasmid with enzymatic digest. there are two enzyme systems:

(1)HindIII singal enzyme

(2)BamHI, XbaI double enzymes

5. Test the result of enzymatic digest with gel electrophoresis. The result isn’t the one we want. Ligation failed

01-08-01

The third time of ligation.

1. Digest the plasmid YIPlac211-E and ADHI promoter with XbalI and BamHI. The digest system is showed below:

YIPlac211-E:

DNA 3ul

Buffer BamHI 4ul

XbaI 0.4ul

BamHI 1ul

ddH2O 31.5ul

total 40 ul

ASHI:

DNA 4ul

Buffer BamHI 6ul

XbaI 0.5ul

BamHI 1ul

ddH2O 48.5ul

total 60 ul

2. Ligation. The carrier(YIPlac211-E) and ADHI is mixed by 1:1.5, 1:3, 1:5.5, respectively, the volume of ligation system is 20μl. Ligation lasts for 3h at 21℃

3. Transform the ligation product into E.coli Top10

3. Cultivate the transformant on LB solid media with 100μg/ml Amp overnight at 37℃

07-08-02

Digest the carrier plasmid YIPlac211-E and plasmid which contains extrogenous fragment ADHIP with XbaI and BamHI overnight at 37℃

07-08-03

1. Do gel electrophoresis with the product of enzymatic digest and recycle the carrier and extrogenous fragment by Gel Extraction Purification

2. Purify the recycled product

(1) mix the product with 1/10 fold NaAc and 2/3 fold pre-colded absolute ethanol and keep it in -20oC for 30 min

(2) centrifuge at 13000rpm. Abandon the supernatant

(3) wash it with 100ul absolute ethanol and centrifuge at 13000rpm, abandon the supernatant. Dry it at 37oC for 15min with it cover uncovered

(4) dissolve it with 10ulTE and conserve it at -20oC

3. Ligation: the proportions of carrier and extrogenous fragment are: 1:1, 1:3, 1:6. the volume of ligation system is 20μl, 21℃, 3h. The ligation system is showed below:

1（ul） 2（ul） 3（ul）

YIPlac211-E 1 1 1

ADHI-E 1 3 6

T4 Buffer 2 2 2

ddH2O 16 14 11

T4ligase 0.4 0.4 0.4

4. Transform the products of ligation into E.coli TOP10, respectively.

5. Cultivate the transferred strain on LB solid media with 100μg/ml Amp for 16h at 37℃.

07-08-04

1. Observe the result of cultivation. There are one transformant on 1:3 and 1:6 plate, respectively

2. Culture these two transformants in LB liquid media with 100μg/ml Amp for ampliative cultivation for 12h at 37℃

3. Distill the plasmid

4. Test the distilled plasmid with enzymatic digest. there are two enzyme systems:

(1)HindIII singal enzyme

(2)BamHI, XbaI double enzymes

5. Test the result of enzymatic digest with gel electrophoresis. The result isn’t the one we want. Ligation failed

07-08-05

The forth time of ligation.

1. Digest the plasmid YIPlac211-E and ADHI promoter with XbalI and BamHI. The digest system is showed below:

YIPlac211-E:

DNA 3ul

Buffer BamHI 4ul

XbaI 0.2ul

BamHI 0.8ul

ddH2O 32ul

total 40 ul

ASHI:

DNA 8ul

Buffer BamHI 6ul

XbaI 0.2ul

BamHI 0.8ul

ddH2O 45ul

total 60 ul

2. Ligation. The carrier(YIPlac211-E) and ADHI is mixed by 1:1 1:3, 1:5, respectively, the volume of ligation system is 20μl. Ligation lasts for 3h at 21℃

3. Transform the ligation product into E.coli Top10

3. Cultivate the transformant on LB solid media with 100μg/ml Amp overnight at 37℃

07-08-06

Digest the carrier plasmid YIPlac211-E and plasmid which contains extrogenous fragment ADHIP with XbaI and BamHI overnight at 37℃

07-08-07

1. Do gel electrophoresis with the product of enzymatic digest and recycle the carrier and extrogenous fragment by Gel Extraction Purification

2. Purify the recycled product

(1) mix the product with 1/10 fold NaAc and 2/3 fold pre-colded absolute ethanol and keep it in -20oC for 30 min

(2) centrifuge at 13000rpm. Abandon the supernatant

(3) wash it with 100ul absolute ethanol and centrifuge at 13000rpm, abandon the supernatant. Dry it at 37oC for 15min with it cover uncovered

(4) dissolve it with 10ulTE and conserve it at -20oC

3. Ligation: the proportions of carrier and extrogenous fragment are: 1:1, 1:3, 1:6. the volume of ligation system is 20μl, 21℃, 3h. The ligation system is showed below:

1（ul） 2（ul） 3（ul）

YIPlac211-E 1 1 1

ADHI-E 1 3 6

T4 Buffer 2 2 2

ddH2O 16 14 11

T4ligase 0.4 0.4 0.4

4. Transform the products of ligation into E.coli TOP10, respectively.

5. Cultivate the transferred strain on LB solid media with 100μg/ml Amp for 16h at 37℃.

07-08-08

1. Observe the result of cultivation. There are one transformant on 1:3 and 1:6 plate, respectively

2. Culture these two transformants in LB liquid media with 100μg/ml Amp for ampliative cultivation for 12h at 37℃

3. Distill the plasmid

4. Test the distilled plasmid with enzymatic digest. there are two enzyme systems:

(1)HindIII singal enzyme

(2)BamHI, XbaI double enzymes

5. Test the result of enzymatic digest with gel electrophoresis. The result isn’t the one we want. Ligation failed