Team:OUC-China/Result/Puf2011d

From 2011.igem.org

Latest revision as of 14:49, 28 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:

LeuB has the correct length at 1000bp, it can be suggested that the second stripe is the vector. Any way, there is no stripe showing around 2000bp compared with the marker,Then we sequenced it, the result can be found here.

2. Ligation of device 2----detecting problem of S01010(16)
        It is quite frustrating when we finally finished ligating device 2 to find it is S01010’s length fault, it directly lead to delay and incomplete re-ligation of device 2. We just regretted that we had been so careless to neglect part quality examination earlier.
        This is an experiment for 18-3&11-16 ligation. 18-3 was cut by EcoRI and SpeI, 11-16 was cut by XbaI and PstI
        Ligation: 18-3(supposed to be 990) 1-16(supposed to be 1788) pSB1AK3(3189)







Lane 4: 11-16 cutting result: have only one stripe at about 3000bps. I supposed that the 11-16 mix with the plasmid, so it should be 3000bps or so.

Lane5: 11-16 plasmid: linearized plasmid pSB1AC3(upper stripe, supposed to be 3055) is about 7000bps long, equals to length of pSB1AK3 plus more than 3000bps.

Checking previous gel record, 11 does not have length problem