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Team:Washington/Protocols/pGA
From 2011.igem.org
(Difference between revisions)
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| - | = | + | =Gibson assembly efficiency assay= |
| - | + | '''Note: Prepare these mixtures on ice''' | |
1. Obtain a 40 uL aliquot of BL21 cells. | 1. Obtain a 40 uL aliquot of BL21 cells. | ||
2. Add 120 uL of ice water to the aliquot. | 2. Add 120 uL of ice water to the aliquot. | ||
| Line 10: | Line 10: | ||
[[File:Washington_iGEM2011_pGAprotocol.png|thumb|right|175px| pGA vector Assembly ]] | [[File:Washington_iGEM2011_pGAprotocol.png|thumb|right|175px| pGA vector Assembly ]] | ||
4. * INS + BCK tubes (x 2) | 4. * INS + BCK tubes (x 2) | ||
| - | + | * add 1 uL of 10X-diluted pGA Gibson product (1A3 BCK/INS pLacGFP) | |
5. * INSctrl tube | 5. * INSctrl tube | ||
| - | + | * add 100 pg of pLacGFP gel extract | |
6. * BCKctrl | 6. * BCKctrl | ||
| - | + | * add 100 pg of 1A3 gel extract | |
| - | + | <br/> | |
| + | Repeat the process for the comparison pSB vector as follows: | ||
[[File:Washington_iGEM2011_pSBprotocol.png|thumb|right|175px| pSB vector Assembly ]] | [[File:Washington_iGEM2011_pSBprotocol.png|thumb|right|175px| pSB vector Assembly ]] | ||
#* 1 uL of pSB Gibson product (T19-1A3/pLacGFP) | #* 1 uL of pSB Gibson product (T19-1A3/pLacGFP) | ||
Revision as of 19:23, 28 September 2011
Gibson assembly efficiency assay
Note: Prepare these mixtures on ice 1. Obtain a 40 uL aliquot of BL21 cells. 2. Add 120 uL of ice water to the aliquot. 3. The total volume is now 160 uL; split evenly among four labeled tubes: INS + BCK, INS + BCK, INSctrl, BCKctrl.
4. * INS + BCK tubes (x 2)
* add 1 uL of 10X-diluted pGA Gibson product (1A3 BCK/INS pLacGFP)
5. * INSctrl tube
* add 100 pg of pLacGFP gel extract
6. * BCKctrl
* add 100 pg of 1A3 gel extract
Repeat the process for the comparison pSB vector as follows:
- 1 uL of pSB Gibson product (T19-1A3/pLacGFP)
- 1 ng INS (pLacGFP- gel extract)
- 1 ng BCK (T19-1A3- gel extract)
- Once all the samples are ready, begin the transformation.
- Rescue each sample in 500 mLs of LB
- Incubate all samples @ 37oC for ~45 min.
- Plate 50 uL on each sample onto a LB + Chlor + Amp + IPTG plate.
- 1 plate for each control in each vector set.
- 3 plates for each Gibson product tube in each set. ( 6 plates for each vector set + 2 control plates)
