IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/07/07
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Yesterday, we ended by transforming pKannabil and pHannibal into E. coli and running a diagnostic digest of digested allergen panel. Yesterday's plates did not grow, and the diagnostic digest did not have good results.
First thing tomorrow, we will culture E. coli containing plasmids for our allergen panel to obtain more plasmids.
For pKannabil and pHannibal:
For Allergen panel
For pKannabil and pHannibal
Transformation of pHannibal pKannibal
This transformation we performed slightly differently from the one yesterday. This protocol is nicknamed the "quick and dirty transform." The transformation we started yesterday of pH and pK didn't work (no growth on either plate).
Quick and Dirty Transform:
PCR of PDK intron
Annealing Temp: 69C Extension Time: 30 sec
Concentrations: PDK:46.5 ng/uL & 36.5 ng/uL Didn't work. Our theory is that the Tm we used (the one IDT sent us) was too high. IDT assumes that the entire primer is going to anneal to a strand of DNA when they calculate their Tm, but since half of our primer won't anneal (the half that doesn't anneal was used to prepend/postpend the biobrick sequences) the Tm IDT calculates will be too high. Here are more correct calculations from Primer3:
OLIGO start len tm gc% any 3' seq LEFT PRIMER 4229 20 51.11 30.00 8.00 3.00 CCAATTGGTAAGGAAATAAT RIGHT PRIMER 5021 20 61.95 45.00 6.00 0.00 TTCGAACCCAATTTCCCAAC
Annealing Temps: 50,55,60,65
Extension Time: 30 sec
For Allergen panel
Digest of Plasmids containing allergen inserts
Digested with Xba and Pst using Fast Fermentas digest protocol. Added 3 μL of DNA and 17μL of master mix (with 0.5μL of each restriction enzyme) to each reaction.
Out of the 24 lanes, 6 were successful. Of the 8 allergens, 3 were successful. Samples of LTP sense, Betv1 sense, and Ger sense had one band at around 3kbps and one band at 300bps. The other genes had one band at 3kbps and additional longer bands on the scale of twice or three times the length of the plasmid.
For the allergens that did not work, we will go back to our bacteria plates and sample 5 additional colonies for each allergen, grow up 10 mL overnight cultures, and miniprep so that we can redo the diagnostic digest. We are hoping that a larger sample size would allow us to find colonies with the correct plasmid.
Miniprep of Barastar and NLS Ligation products
Performed miniprep of overnight cell cultures of 7 colonies of Barstar, 3 colonies of NLS.
Minipreps performed according to pages 22-23 of the QIAprep Miniprep Handbook.
Digestion of Barstar and NLS Ligation products
7 reactions of Barstar and 3 reactions of NLS
In each reaction:
Ran on a 2% agarose E-gel
Lane 1: Ladder
Lane 2-8: Barstar
Lane 9-11: NLS
PCR of LacIN
3 reactions, each with:
1= 95°C for 10:10
2= 95°C for :15
3= 50°C for :30
4= 72°C for 1:30
5= goto 2, 29 times
6=72°C for 10:10
Gel Image of LacIN PCR result:
PCR of Barnase and Gal4DBD
5 reactions each of Barnase from pMT413, Barnase from pMT1002, and Gal4
In pMT413 Barnase reactions:
In pMT1002 Barnase Reactions:
In Gal4DBD reactions:
V0120 Plasmids containing the pENTCUP2 plant promoter, NOS terminator and NOS terminator + STOP were sent to GENEWIZ for sequencing. Sequencing results are expected tomorrow.
5 mL cultures were started from the YFP-2x construct from yesterday, as well as the B15 (StrepII) tag.
Cultures were placed in a 37°C incubator and left to shake overnight.
Primers for Wintergreen pathway parts