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Team:Washington/Protocols/pGA
From 2011.igem.org
(Difference between revisions)
| Line 8: | Line 8: | ||
# Add 120 uL of ice water to the aliquot. | # Add 120 uL of ice water to the aliquot. | ||
# The total volume is now 160 uL; split evenly among four labeled tubes: INS + BCK, INS + BCK, INSctrl, BCKctrl. | # The total volume is now 160 uL; split evenly among four labeled tubes: INS + BCK, INS + BCK, INSctrl, BCKctrl. | ||
| + | [[File:Washington_iGEM2011_pGAprotocol.png|thumb|right|175px| pGA vector Assembly ]] | ||
#* INS + BCK tubes (x 2) | #* INS + BCK tubes (x 2) | ||
#** add 1 uL of 10X pGA Gibson product (1A3 BCK/INS pLacGFP) | #** add 1 uL of 10X pGA Gibson product (1A3 BCK/INS pLacGFP) | ||
| Line 15: | Line 16: | ||
#** add 100 pg of 1A3 gel extract | #** add 100 pg of 1A3 gel extract | ||
#Repeat steps 1-3 for the comparison vector pSB using the following instead: | #Repeat steps 1-3 for the comparison vector pSB using the following instead: | ||
| + | [[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) | ||
#* 1 ng INS (pLacGFP- gel extract) | #* 1 ng INS (pLacGFP- gel extract) | ||
Revision as of 22:16, 27 September 2011
pGA vector Assay
- Prepare these mixtures on ice
- Obtain a 40 uL aliquot of BL21 cells.
- Add 120 uL of ice water to the aliquot.
- The total volume is now 160 uL; split evenly among four labeled tubes: INS + BCK, INS + BCK, INSctrl, BCKctrl.
- INS + BCK tubes (x 2)
- add 1 uL of 10X pGA Gibson product (1A3 BCK/INS pLacGFP)
- INSctrl tube
- add 100 pg of pLacGFP gel extract
- BCKctrl
- add 100 pg of 1A3 gel extract
- INS + BCK tubes (x 2)
- Repeat steps 1-3 for the comparison vector pSB using the following instead:
- 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)
