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Team:Hawaii/Ligation of pRL1383a Parts
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Contents |
Ligation of Parts
- The BioBrick parts of pRL1383a are to be ligated in a series of experiments.
Methods
Restriction Digest
- Each part is digested with both enzymes. For all cases NEBuffer 2 is used.
- Reaction Conditions:
- 5ul Buffer
- 1ul Enzyme 1 + 1ul Enzyme 2
- 0.5 ul BSA
- Xul water
- Yul insert (1ug if possible)
- Zul vector (less than 1ug)
- Running Conditions: 2 hours at 37°C.
- Check the progress of the reaction by running a 0.8% gel.
Ligation
- Refer to http://openwetware.org/wiki/DNA_ligation for the reasoning behind this.
Transformation
Verification with Colony PCR
Results
7/27
- A few re-digests and ligations were performed today: oriT + pSB1A3, oriV + pSB1A3, mob + B0024, rep + R0010, aadA(pRL1383a+R0010). Afterwards, the ligation product was transformed to DH5-alpha. Only OriT transformed. I did not check the progress of this experiment with a gel, so I am not sure where this went wrong.
- Next time, a gel will be run after every step.
| Name | size | enzyme | quantity |
|---|---|---|---|
| oriT | ~125bp | XbaI & PstI | n/a |
8/11
| Name | size | enzyme | quantity |
|---|---|---|---|
| rep | 3.3kb | XbaI & PstI | 5ng/ul |
| oriV | 415bp | XbaI & PstI | 5ng/ul |
| aadA (pRL1383a) | 806bp | XbaI & PstI | 5ng/ul |
| aadA (BB) | 806bp | XbaI & PstI | 5ng/ul |
| P1 lytic Region | 1.3kb | EcoRI & SpeI | 5ng/ul |
| BBa_B0030 | 2094bp | SpeI & PstI | 4ng/ul |
| BBa_B0015 | 3318bp | EcoRI & XbaI | 5.7ng/ul |
| pSB1A2 | 2094bp | SpeI & PstI | 5.5ng/ul |
| Name | strain, antibiotics | colonies? after next day??? | PCR verification in bp theoretical, experimental |
|---|---|---|---|
| rep(20ng)+B0030(16ng) | DH5-alpha & Amp100 | lawn | 3.3kb, 300&600 |
| oriV(20ng)+pSB1A2(50ng) | DH5-alpha & Amp100 | 21 colonies | 676, 600 |
| aadA (BB)+ B0030 | DH5-alpha & Amp100 | lawn | 900, #4:900, others: 300 |
| aadA(pRL1383a)+B0030 | DH5-alpha & Amp100 | lawn | 900, 300 |
| P1 lytic + B0015 | DH5-alpha & Kan50 | no colonies | n/a |
Transformation & Verification PCR
- The only bands corresponding to the correct size are the 4 oriVs and the 4th BB version of aadA.
- The rep region lanes are all either 300 or 600bp when they should be 3.3kb. If the RBS (B0030) ligated back to itself, the band should be 253bp. The bands at 300bp may be explained by this. It is unclear what happened in the latter case. The incorrect aadA bands are also around 300bp which would mean the RBS containing vector ligated back to itself some how. B0030 was cut with SpeI and PstI.
Plasmid Prep
- OriV1-4 and aadA (BB) were cultured over-night in Terrific Broth with amp100 as selection.
- The plasmids were isolated using an Alkaline Lysis Mini-Prep.
8/13
- Three ligations were performed today. I checked my math from the last ligation and decided it wasn't totally correct for the rep ligation to B0030.
- P1 lytic was ligated to B0015 (TT), Rep to B0030 with precisely 2:1 insert to vector ratio, and the aadA region from pRL1383a to B0030.
- Unfortunately there is no gel to confirm this ligation.
Transformation
- The ligation products were transformed into DH5-alpha cells with selection on LB amp100 plates. pUC18 was transformed into DH5-alpha cells as a positive control, while no plasmid was used in a transformation as a negative control.
- A lawn of colonies was observed for all three transformations. No colonies grew for either control (need to check pUC18 and see if there are problems with our stock).
PCR Verification
Discussion
- What was learned and how to do future experiments differently.
