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Results
We ran a 1% gel of the digest before and after purification. We had a decent yield, maybe 40% of our initial digest product in the purified lanes.
Team:Genspace/parts
From 2014.igem.org
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Project
What is the Open Lab?
Translational_Unit
Part:BBa_K1429002
Designed by: Christal Gordon Group: iGEM14_Genspace (2014-09-29)
Cyan Fluorescent Protein (CFP) "Cindy Lou" coding region, intellectual property-free
IP free cyan fluorescent protein. We have a ribosome binding site (RBS).
Background
We want to take Tuesday's PCR products and put them into the pSB1C3 backbone.
Digest PCRs:
10 ul PCR product
2 ul cutsmart buffer (10x stock)
1 ul PstI
1 ul EcoRI
20 ul total --> incubate for 30 min at 37C
PCR purify digest product (only 14 ul - save 6 ul):
Follow kit protocol. Elute in elution buffer.
Worried that the washed columns won't bind DNA, we are going to use some of the set-aside (unpurified) digest product for a backup ligation. We'll run a gel of our purification, but we are going to set up a ligation beforehand, so we won't have even rough estimates of DNA concentrations.
Set up ligations:
| Component | Using purified digest product | Using unpurified digest product | BB alone |
| dH2O | x | 11 | 14 |
| Insert (RFP or GFP) | 14 | 3 | x |
| 1:10 BB | 3 | 3 | 3 |
| T4 buffer (10 | 1 | 1 | 1 |
The above were incubated 30 min at RT then stored at -20C.
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Conclusions
Next step: Transform the ligated plasmids into E. coli!
