1128 pDB.His.PfuMBP-TEV

Cloning of PfuMBP by Gene synthesis

5/1/2012

PCR 2 fragments, 25 cycles using Phusion or Kapa HiFi, cut out fragments and purify by gel extraction kit.

Join two fragments by Gibson

mix two PCR fragments, and join them using Gibson Mix

purify, jointed fragment with Nde1 + Kpn1, cut the top band,  digest pDB.His.MBP with Nde1 + Kpn1, ligate and transform DH5lapha.

5/3/2012: 

5/5/2012, make more miniprep digest with Nde1 + Kpn1

digest miniprep with Kpn1+ Nde1. DNA marker is Fermentas 1kb plus (20000, 10000, 7000, 5000, 4000, 3000, 2000, 1500, 1000, 700, 500, 400, 300, 200, 75). size is too big (should be 1180bp) so the top band is not the right product, should use the bottom band.

Select 1128-2 to sequence (result is not good, poor quality)

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REPEAT

5/5/2012, repeat gene synthesis and cloning, using Kapa HiFi.

gene synthesis:

fragment 1, 10 cycles with 0.25 ul Kapa HiFi without outer primers

Fragment 2, 10 cycles with 0.25 ul Kapa HiFi without outer primers

Mix reaction 1 + 2, 20 cycles

digest gel purified fragment with Nde1 + Kpn1, digest vector 360 with Nde1 + Kpn1

5/6/2012

ligate 4 hours and transform DH5alpha

1128 PCR Screening 5/7/2012

Select 4 clones to mini-prep

1128 plasmids are digested by Nde1+Kpn1

sequencing: 2 and 4 have errors

Cloning of 1128 pDB.His.PfuMBP-TEV

Purpose: Cloning of 1128 pDB.His.PfuMBP-TEV

Date: 2012-5-16---2012-5-22

Notebook:0

Page:

Clone ID:1128

Experimenters: yingying

Results

cloning

1. PCR 5 cycles 0.5 ul of Pfu-MBP1~Pfu-MBP1-16 mix, 5 cycles 0.5 ul Pfu-MBP2-1 ~Pfu-MBP2-16 mix, in 25 ul final volume.
2. Mix reaction 1 + 2, add 1 ul of 10 uM Pfu-MBP1-1 & 1 ul of 10 uM Pfu-MBP2-16, PCR 25 cycles.

	band 1	band 2
	PCR product	360 pDB.His.MBP
enzymes	Kpn1 + Nde1

2012-5-18

	band 1	band 2
	PCR product	360 pDB.His.MBP
enzymes	Kpn1 + Nde1

Colony Screening

Date: 2012-5-21

Make miniprep and digest with Kpn1 + Nde1

clone 2 need to sequence

Result intepretation/Summary

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Conclusion

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Cloning of 1128 pDB.His.PfuMBP-TEV

Purpose: Cloning of1128 pDB.His.PfuMBP-TEV

Date: 2012-5-22---2012-5-25

Notebook:0

Page:

Clone ID:1128

Experimenters: yingying

Results

cloning

Template: 1129 pDB.His.PfuMBP

1 ul of 10 uM Pfu-MBP1-1 & 1 ul of 10 uM Pfu-MBP2-16, PCR 25 cycles

	band 1	band 2
	PCR product	360 pDB.His.MBP
enzymes	Kpn1 + Nde1

Colony Screening

Date: 2012-5-25

Make miniprep and digest with Kpn1 + Nde1

clone 1,2 may be right

Result intepretation/Summary

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Conclusion

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