Minnesota/24 June 2008

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(Difference between revisions)
 
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|1. '''Gel Electrophoresis:''' Used this technique to show plasmid DNA sequence. Materials:
|1. '''Gel Electrophoresis:''' Used this technique to show plasmid DNA sequence. Materials:
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|a. 50 uL of 1% agarose gel
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|'''a.''' 50 uL of 1% agarose gel
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|b. TAE Buffer
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|'''b.''' TAE Buffer
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|c. One gram of 1% agarose per 100 uL of TAE
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|'''c.''' One gram of 1% agarose per 100 uL of TAE
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|d. Ethidium bromide (intercalating agent)
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|'''d.''' Ethidium bromide (intercalating agent)
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|'''Problem Encountered:''' electrophoretic gels with 1% agarose had deficient wells
|'''Problem Encountered:''' electrophoretic gels with 1% agarose had deficient wells
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|2. '''Plating''' from 6-23-08 transformations again.
|2. '''Plating''' from 6-23-08 transformations again.
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|a. Since plating of the 6-23 transformations provided no colonies for parts 15-18, the remaining cells from those transformations were re-plated. 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.
+
|'''a.''' Since plating of the 6-23 transformations provided no colonies for parts 15-18, the remaining cells from those transformations were re-plated. 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.
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|b. Plates were placed at 37C in an incubator and allowed to grow overnight.  
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|'''b.''' Plates were placed at 37C in an incubator and allowed to grow overnight.  
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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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{|
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|a. All primers were spun down prior to opening. The appropriate amount of water was added to resuspend each primer in solutions of 100 ug/uL and 10 ug/uL. The 10 ug/uL solution is our working concentration. Primers were then stored at -20C.  
+
|'''a.''' All primers were spun down prior to opening. The appropriate amount of water was added to resuspend each primer in solutions of 100 ug/uL and 10 ug/uL. The 10 ug/uL solution is our working concentration. Primers were then stored at -20C.  
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|b. 12 uL reactions containing plasmid to be sequenced and the corresponding primer(s) were set up and submitted for sequencing. Each reaction mixture contained 1 ul primer, 1 ul plasmid DNA, and 10 uL dd H2O. All samples were sent for sequencing at the Biomedical Genomics Center at the U of M.  
+
|'''b.''' 12 uL reactions containing plasmid to be sequenced and the corresponding primer(s) were set up and submitted for sequencing. Each reaction mixture contained 1 ul primer, 1 ul plasmid DNA, and 10 uL dd H2O. All samples were sent for sequencing at the Biomedical Genomics Center at the U of M.  
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Latest revision as of 21:17, 8 July 2008

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1. Gel Electrophoresis: Used this technique to show plasmid DNA sequence. Materials:
a. 50 uL of 1% agarose gel
b. TAE Buffer
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 re-plated. 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 prior to opening. The appropriate amount of water was added to resuspend each primer in solutions of 100 ug/uL and 10 ug/uL. The 10 ug/uL solution is our working concentration. Primers were then stored at -20C.
b. 12 uL reactions containing plasmid to be sequenced and the corresponding primer(s) were set up and submitted for sequencing. Each reaction mixture contained 1 ul primer, 1 ul plasmid DNA, and 10 uL dd H2O. All samples were sent for sequencing at the Biomedical Genomics Center at the U of M.
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