Minnesota/7 July 2008

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Latest revision as of 21:18, 23 July 2008

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1. Spectophotometry: 'Spec' MCherry. Want to measure DNA concentration in MCherry solutions.

2. Gel Electrophoresis: Run PCR, MCherry samples in 0.8% agarose gel to see if correct DNA is present.

3. Simulations: Begin simulations on SynbioSS by using model reaction network.

4. PROBLEM: Gels under UV light show no PCR DNA bands. Must redo PCR and run through another gel.

5. Double Digests: Digest Terminator, base vector, and promoter. Run through gel to check size.

6. Meeting with Yiannis: 1pm meeting with grad students and advisor. Discussed:

a. Received DB3.1 competent E. Coli cells. Transformed base vectors containing ccdB gene into DB3.1 cells that are resistant to the lethal ccdB gene.

b. Transformed TOP10 cells to the DH5alpha cells because want all DNA to be inside same cells. Problem resolved - DH5alpha cells are fine, but the amount of DNA sent in iGEM notebook was too little.

c. Made glycerol stocks.

d. Started PCR. PCR cycle copies not only the section of DNA that want copied but also copies RBS on 5' end. Then after copied DNA sections with RBS are made, then will add restriction site enzymes that cut the copied DNA and RBS section at the appropriate restriction site. NOTE: PCR did not show up in gel, must redo.

e. Need to do: (1) Ligation - need to insert promoter-gene-terminator into base vector. Will be blunt-end ligation because base vector and everything else has been cut by restriction site enzymes so has a blunt-end to ligate. (2) Simulations - finish simulations on SynbioSS. (3) Range of colors - think about range of colors and color genes. (4) ***Model*** HAVE TO DO THIS so have sense of what should expect when running experiments and will help guide the rest of the project.

@@ Line 16: / Line 16: @@
 |4. '''PROBLEM:''' Gels under UV light show no PCR DNA bands. Must redo PCR and run through another gel.
 |-
-|5. '''Meeting with Yiannis''': 1pm meeting with grad students and advisor. Discussed:
+|5. '''Double Digests''': Digest Terminator, base vector, and promoter. Run through gel to check size.
 |-
-| '''a.''' Received DB3.1 competent E. Coli cells. Transformed base vectors into DB3.1 cells.
+|[[Image:digest.JPG|600px||center|thumb|Digest diagram]]
+|-
+|6. '''Meeting with Yiannis''': 1pm meeting with grad students and advisor. Discussed:
+|-
+| '''a.''' Received DB3.1 competent E. Coli cells. Transformed base vectors containing ccdB gene into DB3.1 cells that are resistant to the lethal ccdB gene.
 |-
 |'''b.''' Transformed TOP10 cells to the DH5alpha cells because want all DNA to be inside same cells. Problem resolved - DH5alpha cells are fine, but the amount of DNA sent in iGEM notebook was too little.
@@ Line 26: / Line 30: @@
 |'''d.''' Started PCR. PCR cycle copies not only the section of DNA that want copied but also copies RBS on 5' end. Then after copied DNA sections with RBS are made, then will add restriction site enzymes that cut the copied DNA and RBS section at the appropriate restriction site. NOTE: PCR did not show up in gel, must redo.
 |-
-|'''e. Need to do:''' (1)''Ligation'' - need to insert promoter-gene-terminator into base vector. Will be blunt-end ligation because base vector and everything else has been cut by restriction site enzymes so has a blunt-end to ligate. (2) ''Simulations'' - finish simulations on SynbioSS. (3)
+|'''e. Need to do:''' (1) ''Ligation'' - need to insert promoter-gene-terminator into base vector. Will be blunt-end ligation because base vector and everything else has been cut by restriction site enzymes so has a blunt-end to ligate. (2) ''Simulations'' - finish simulations on SynbioSS. (3) ''Range of colors'' - think about range of colors and color genes. (4) ''***Model***'' HAVE TO DO THIS so have sense of what should expect when running experiments and will help guide the rest of the project.
 |}