User:Elizabeth Ghias/Notebook/Experimental Biological Chemistry/2011/09/20: Difference between revisions
From OpenWetWare
(Autocreate 2011/09/20 Entry for User:Elizabeth_Ghias/Notebook/Experimental_Biological_Chemistry) |
No edit summary |
||
(8 intermediate revisions by the same user not shown) | |||
Line 10: | Line 10: | ||
==Objective== | ==Objective== | ||
To mutate GFP through PCR so that it contains a cysteine residue after the enterokinase cleavage site. | |||
To prepare starter culture media and protein expression culture media for protein expression. | |||
==Description== | ==Description== | ||
<u>PCR</u> | |||
# The PCR quick change manual was used to determine which forward and reverse primers should be used for the PCR. Prepared primers were used for the PCR reaction | |||
# A reaction mixture was prepared with the following contents: 1 μL of template DNA, 1.25 μL of primer 1 (D1C F), 1.25 μL of primer 2 (D1C R), 5 μL of 10X Pfu reaction buffer, 1 μL of dNTP mix, and 1 μL of PfuTurbo DNA polymerase. | |||
# 25 μL of Bio Rad Chill Out Wax was placed on top of the reaction mixture | |||
# The reaction mixture was placed in the thermocycler for one cycle at 95°C for 30 seconds | |||
# The reaction mixture was kept in the thermocylcer for 16 cycles with the profile shown below. | |||
# The reaction mixture was left in the thermocycler for 24 hours at 4°C | |||
PCR Profile | |||
1) 95 °C for 30 seconds | |||
2) 55°C for 1 minute | |||
3) 68°C fr 3.6 minutes | |||
<u>Protein Expression Preparation</u> | |||
Starter Culture Media: | |||
# In a 250 mL flask, 0.875 g of Lysogeny (Luria) Broth and 35 mL of water were mixed. Four flasks were prepared with this mixture. | |||
# The flasks were covered in foil and autoclaved for 50 minutes at 121 F. | |||
# Once the flasks cooled, 35 μL of 1000X ampicillin antibiotic was added to each flask. The ampicillin was prepared by mixing 0.450 g ampicillin with 4.5 mL H<sub>2</sub>O. | |||
# The flasks were then inoculated with a single colony of bacteria using sterile methods. | |||
# The flasks were placed in a incubator/shaker set to 37°C and 200 rpm. The flasks were left in the shaker/incubator overnight. | |||
Expression Culture Media: | |||
# In a 2.8 L Fernback flask, 25G or Luria Broth and 1 L of water were mixed. Four flasks were prepared in this manner. | |||
# The flasks were covered in foil and autoclaved for 50 minutes at 121 F. | |||
# 1000X antibiotic was prepared by mixing 0.450 g ampicilin 4.50 mL of water. | |||
# Once the flasks had cooled, 1 mL of 1000X antibiotic was added to each of the four flasks. | |||
# 4 mL of 0.1 M isopropyl β-D-1 thiogalactopyranoside (IPGT)was also prepared for use the following day. | |||
==Data== | ==Data== |
Revision as of 13:54, 27 September 2011
Experimental Biological Chemistry | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> |
ObjectiveTo mutate GFP through PCR so that it contains a cysteine residue after the enterokinase cleavage site. To prepare starter culture media and protein expression culture media for protein expression. DescriptionPCR
PCR Profile 1) 95 °C for 30 seconds 2) 55°C for 1 minute 3) 68°C fr 3.6 minutes
Starter Culture Media:
Expression Culture Media:
Data
NotesThis area is for any observations or conclusions that you would like to note.
|