Minnesota/24 June 2008

From 2008.igem.org

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|3. '''Sequencing primers''' ordered on 6-20-08 were picked up. All primers were diluted to mircomoles according to the following additions of sterile H20:
|3. '''Sequencing primers''' ordered on 6-20-08 were picked up. All primers were diluted to mircomoles according to the following additions of sterile H20:
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{|border="1"
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!| ||Primer ||nmoles    ||uL H20 added
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| ||P22 cII cR ||37.7      ||377.0
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|-
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| ||P22 cII cF ||36.0      ||360.0
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|-
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| ||Lambda cI R ||32.40    ||324.0
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|-
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| ||Lambda cI F ||33.2      ||332.0
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|-
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| ||P22 MNT R ||32.8      ||328.0
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|-
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| ||P22 MNT F ||28.2      ||282.0
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|-
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| ||EYFP R ||43.8      ||438.0
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|-
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| ||EYFP F      ||34.5      ||345.0
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|-
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| ||pSB 2K3    ||39.6      ||396.0
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|-
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| ||pSB 1A2    ||31.7      ||317.0
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|-
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| ||pSB 1AK3    ||40.0      ||400.0
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| ||GFP R      ||32.1      ||321.0
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| ||GFP F      ||33.7      ||337.0
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| ||mCherry R  ||43.9      ||439.0
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| ||mCherry F  ||28.4      ||284.0
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| ||LacI R      ||29.4      ||294.0
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| ||LacI F      ||31.2      ||312.0
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|}
|a. All primers were spun down before being opened and H20 added. Primers were then stored at -20C.  
|a. All primers were spun down before being opened and H20 added. Primers were then stored at -20C.  
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|b. 6 uL reactions containing plasmid to be sequenced and the corresponding primer(s) were set up and submitted for sequencing.
|b. 6 uL reactions containing plasmid to be sequenced and the corresponding primer(s) were set up and submitted for sequencing.

Revision as of 15:20, 1 July 2008

1. Gel Electrophoresis: Used this technique to show plasmid DNA sequence. Materials:
a. 50 uL of 1% agarose gel
b. Buffer TAE
c. One gram of 1% agarose per 100 uL of TAE
d. Ethidium bromide (intercalating agent)
Problem encountered: electrophoretic gels with 1% agarose had deficient wells
Solution: add 0.5 grams more of agarose to the 100uL of TAE buffer


Electrophoretic gel run on 6/24
2. Plating from 6-23-08 transformations again.
a. Since plating of the 6-23 transformations provided no colonies for parts 15-18, the remaining cells from those transformations were replated. 75 uL of cell culture was spread on each of two plates for each culture; plates contained LB media and the corresponding antibiotic. A metal spreading tool was used to spread the culture suspension on the plates, and this was sterilized between each sample by dipping it in 100% ethanol (EtOH) and flaming it. 75 uL cell culture was pipetted on, and spread around plate.
b. Plates were placed at 37C in an incubator and allowed to grow overnight.
3. Sequencing primers ordered on 6-20-08 were picked up. All primers were diluted to mircomoles according to the following additions of sterile H20:


Primer nmoles uL H20 added
P22 cII cR 37.7 377.0
P22 cII cF 36.0 360.0
Lambda cI R 32.40 324.0
Lambda cI F 33.2 332.0
P22 MNT R 32.8 328.0
P22 MNT F 28.2 282.0
EYFP R 43.8 438.0
EYFP F 34.5 345.0
pSB 2K3 39.6 396.0
pSB 1A2 31.7 317.0
pSB 1AK3 40.0 400.0
GFP R 32.1 321.0
GFP F 33.7 337.0
mCherry R 43.9 439.0
mCherry F 28.4 284.0
LacI R 29.4 294.0
LacI F 31.2 312.0
a. All primers were spun down before being opened and H20 added. Primers were then stored at -20C.
b. 6 uL reactions containing plasmid to be sequenced and the corresponding primer(s) were set up and submitted for sequencing.
Retrieved from "http://2008.igem.org/Minnesota/24_June_2008"