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From 2011.igem.org

9-19

Enlargement culture the 6&leu ligated in 3A method at 11 a.m. and coat them on the square plates then cultured in incubator.

After electrophoresis of the no.6 and the carrier C and cutting the gels, we recover them in gel extraction mini kit. at 12 a.m. The carrier C is uesd for standardization.

Put E0020,C0076 and praiI in LB again. Disinfect 7 test tubes.

We side the concentration of recovered 6 and leu. But they are too slow to be concentrated. After being concentrated, no.6’s concentration has been to 30, but leu is the same. So we prepare to cut Leu again.

In the evening, we side the concentration of 13 parts which have been standardized yesterday.

9-20

Ligating 6&leu in 3A method failed. Leu are double-enzyme cutd to be ligated with no.6 in traditional method.

C0076,E0020 and praiI are preservated and isolated at 1 p.m.

9-21

Make 6&leu which were ligated yesterday heated to be inactivited and transformed and then coat them on square plates.

Put raiR1、raiR2、raiR3、raiI1、raiI2、sinR1、sinR2、sinR3 in EP tubes to shake culturing to be used in sequencing by PCR.

Asparaguspea are cultured in shaking bed at 30℃ waiting for PCR.

Put the primer ordered in EP tubes.

9-22

6&leu’s plates have come out bacteria colonies. Pick out some in LB.

Enzyme cut E0020,C0076 and raiI1,2,3. but we find we use the wrong enzyme. We do it again in the afternoon.

Amplifications by PCR: rhiI、rhiR、rhiABCpro、rhiIpro、pro-RBS-rhiI.

Verify by PCR: raiR1、raiR2、raiR3、raiI1、raiI2、sinR1、sinR2、sinR3. Then verify them in electrophoresis.

9-23

Extract raiI,raiR,raiABCpro. The parts—raiIpro,pro-RBS-raiI which were fail to be synthetized keep on PCR. So does the parts which were verified in PCR.

Extracte the plasmids of 6&leu, and they’re correct verified by enzyme cut.

LB square plates with C+ have lost efficacy. So we make up medium with antibiotics.

Double-enzyme cut with EcoRI and PstI: raiI、raiR、raiABCpro、18-3-11、14-19、circuit-I、5&4、14-19-13 and carrier C.

Throw away some square plates with blended bacteria after being desinfected.

9-24

6&leu have been ligated successfully. From today, we begin to finish the circuit-I,II,III to put the Leu in the circuits.

Enzyme cut 5-4,6&leu,18-3-11,13,carrier AC, carrier AK, C0076 and 6. extracte 6 and carrier AK. Ligate 18-3-11&eu-6；13&leu-6；C0076&6.

Activate the strain of 5-4,3,E0020 from preservation tubes.

Clear up preservation tubes. Throw away raiR2、raiI1、sinR3。SinI1、2、3；psinI1、2. Put raiIpro1,2,3 in EP tubes to be cultured and then be sequenced with sinR1、2；raiI2、raiR1.

PCR rhiI、rhiR、rhiABCpro, rhiIpro、pro-RBS-rhiI.

Standardizations: ligate carrier C separately with circuit-I,18-3-11,14-19,5-4,14-19-13.

9-25

The plates which are used for standardizing 14-19,circuit-I and 18-3-11 aren’t all successful. Only the 14-19 plates have bacteria colony. Pick out the bacteria colony of14-19 and RFP into LB. Circuit-I ,18-3-11 and 14-19 are transformed again. In the afternoon,we also transform some parts-- rhiR、rhiABCpro、5&4、18-3-11&leu-6、13&leu-6、C0076&6, and coat them on the square plates.

Recover the product—rhiI by PCR and ligate it and rhiR, LeuB with carrier C to be standardized.

In the evening, we standardized the parts—leuB,14-19-13,rhiR and RFP.

Extract the plasmids of 5-4, 3 and E0020 activated yesterday.

Ligate E0020 with 6.

We transforme the part-J13002 and coat them on square plates.