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Team:OUC-China/Result/Puf2011d
From 2011.igem.org
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Since the first time we use the linearized plasmid pSB1A2 for part ligation, it has serious quality problems, perhaps it is because its length and times of PCR, maybe that mutation is not avoidable. So we use bacteria to do the plasmid amplification, but unfortunately pSB1C3 from K381001 still has length problem, we just have to transform another part J04450. Much time was wasted because of inexperience.<br> | Since the first time we use the linearized plasmid pSB1A2 for part ligation, it has serious quality problems, perhaps it is because its length and times of PCR, maybe that mutation is not avoidable. So we use bacteria to do the plasmid amplification, but unfortunately pSB1C3 from K381001 still has length problem, we just have to transform another part J04450. Much time was wasted because of inexperience.<br> | ||
The followings are experiments from which we find this problem:<br> | The followings are experiments from which we find this problem:<br> | ||
| + | <b>1. Standardizing LeuB(K594002)----detecting problem with K381001</b></br> | ||
| + | —Two restrict enzyme cutting<br> | ||
| + | K381001,50μL cutting system , use EcoRI and PstI (NEB), 37℃ incubation for 3 hours<br> | ||
| + | leuB Product of PCR, 50μL cutting system , cutting with EcoRI and PstI (NEB), 37℃ incubation for 3 hours<br> | ||
| + | --Gel extraction<br> | ||
| + | --Ligation: add 8 μl pSB1C3 from K381001, 9μLleuB， 2μL 10×T4 ligase buffer， 1 μL T4 ligase.16℃ 8 hours<br> | ||
| + | --Transform into DH5α，liquid LB medium amplification， extract plasmid， run two-enzyme cutting， run a 1% agarose electrophoresis， the gel map is showing as follows:<br> | ||
Revision as of 03:06, 5 October 2011
Note:
1.This form is quite useful for laboratory part or plasmid information tracking, such as analyze gel result and examine ligation correctness.
2.Through our experiment, all parts have its specific 2011-OUC-order for our convenience. In our notebook
, many parts are called in this order number but not its original biobrick BBa_ name. If you have any problem reading our notebook, please refer to this form.
Note:
Since the first time we use the linearized plasmid pSB1A2 for part ligation, it has serious quality problems, perhaps it is because its length and times of PCR, maybe that mutation is not avoidable. So we use bacteria to do the plasmid amplification, but unfortunately pSB1C3 from K381001 still has length problem, we just have to transform another part J04450. Much time was wasted because of inexperience.
The followings are experiments from which we find this problem:
1. Standardizing LeuB(K594002)----detecting problem with K381001
—Two restrict enzyme cutting
K381001,50μL cutting system , use EcoRI and PstI (NEB), 37℃ incubation for 3 hours
leuB Product of PCR, 50μL cutting system , cutting with EcoRI and PstI (NEB), 37℃ incubation for 3 hours
--Gel extraction
--Ligation: add 8 μl pSB1C3 from K381001, 9μLleuB， 2μL 10×T4 ligase buffer， 1 μL T4 ligase.16℃ 8 hours
--Transform into DH5α，liquid LB medium amplification， extract plasmid， run two-enzyme cutting， run a 1% agarose electrophoresis， the gel map is showing as follows:
