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| | : I. DNA purification | | : I. DNA purification |
| - |
| |
| | :: * Put 5 ul of phage sample (S4, S10, S21, L8, and WT) and 45 ul of ddH20 into in PCR tubes with respective labeling. | | :: * Put 5 ul of phage sample (S4, S10, S21, L8, and WT) and 45 ul of ddH20 into in PCR tubes with respective labeling. |
| - |
| |
| | :: * Boil for 12 minutes in the PCR machine (98 Celsius). | | :: * Boil for 12 minutes in the PCR machine (98 Celsius). |
| - |
| |
| | :: * Remove the tubes from the PCR machine and keep on ice until use in PCR. | | :: * Remove the tubes from the PCR machine and keep on ice until use in PCR. |
| | | | |
| - | 2)
| + | : II. PCR w/ Phusion polymerase |
| - | 2) Propagating Mutant Phage (10.10)
| + | :: * To a eppendorf tube, add (for 6 PCR reactions simultaneously): |
| | + | ::: - 210 uL of ddH2O |
| | + | ::: - 60uL 5x Phusion buffer |
| | + | ::: - 9uL 10mM dNTPs |
| | + | ::: - 6uL of each primer (BI309 and BI310) |
| | + | :: * Mix well |
| | + | :: * To six new PCR tubes, labeled S4, S10, S21, L8, and WT respectively, add 2uL of template DNA from the boiled samples. |
| | + | :: * Add 3uL of Phusion Polymerase to the eppendorf tube (master mix). |
| | + | :: * Add 48uL of the master mix into each PCR tube. |
| | + | :: * Run 35 cycles with temperatures of 95 C, 50 C, and 72 C with an extension time of 1.5 minutes. |
| | + | :: * Leave overnight in the freezer. |
| | | | |
| - | * Three different conditions of propagation was used for WT, S4, S10, and L8 | + | : III. Low melt gel electrophoresis (10.19) |
| - | :: - Erlenmeyer flask: 10 mL LB + 1 mL E coli B liquid culture overnight + 10 uL of phage
| + | :: * Add 75 ml of 1X TAE buffer and 0.75 grams of low melt agarose to an Erlenmeyer flask. Put into the microwave for about 90 seconds or until the agarose is completely dissolved. 1 drop of ethidium bromide was then added and mixed with the gel. |
| - | :: - 15 mL centrifuge tube: 5 mL LB + 0.5 mL E coli B liquid culture overnight + 10 uL of phage | + | :: * Pour the liquid onto the gel bed and let it cool. Remember to insert a sample comb. |
| - | :: - autoclaved test tube: 1 mL of LB + 100 uL E coli B liquid culture overnight + 10 uL of phage | + | :: * The gel allowed to sit for overnight in the fridge to solidify. |
| - | | + | :: * Move the gel into the proper orientation (DNA runs towards the red electrode) in the gel box, cover the gel with 1X TAE buffer and load 8 ul of each of the PCR products with 3 ul of loading dye mixed in. Add a DNA ladder as a reference. Turn on the power supply and run at 80 volts. |
| - | * Wild-type and mutant phages were allowed to propagate for approximately 24 hours before purified via centrifugation and choloroform. | + | ::: ''This is the procedure we used for our first round of electrophoresis. Unfortunately, the sample produced will not be enough for ligation (40-50uL of sample is need for loading). Thus we had to repeat this procedure.'' |
| - | | + | |
| - | * Spot tests at -2, -4, -6, and -8 was performed for each sample to estimated phage concentration after propagation. | + | |
| - | | + | |
| - | * Each phage sample was then plated at -7 using x4 agar to verify the mutants' phenotypic stability after propagation.
| + | |
| - | | + | |
| - | 3) TEM (10.16) | + | |
| - | | + | |
| - | * Phage Purification Team set up copper grids and arranged for TEM appointments to look at S4, S10 and L8
| + | |
| - | | + | |
| - | 4) Sequencing (10.17-10.?)
| + | |
| - | | + | |
| - | * During the week when we were waiting for the new sequencing primers (BI319 and BI320) to arrive, we where able to isolate a new mutant phage S21. Thus, we will sequence S4, S10, S21, L8, and WT T7 phage to map out mutations altering phage capsid sizes.
| + | |
| - | | + | |
| - | * DNA isolation, PCR, and gel electrophoresis protocol was similar to that of [[Team:BYU_Provo/Notebook/SmallPhage/Springexp/Period2/Exp/5.20_T7_Minor_Capsid_Protein_PCR| 5.20 T7 Minor Capsid Protein PCR]]
| + | |
| - | | + | |
| - | : ''Note for sequencing we used the primers BI257 and BI258.''
| + | |
| | | | |
| | '''V) Results''' | | '''V) Results''' |
| - |
| |
| - | 2) Propagating Mutant Phage
| |
| - |
| |
| - | * Plaque formed up to -6 for each sample, implying a concentration of at least 10E8 PFU/mL
| |
| - |
| |
| - | * Propagated phage retained their larger or smaller capsid size as confirmed by their smaller and larger plaque sizes at x4 agar.
| |
| - |
| |
| - | 3) TEM
| |
| - | '''ADD PICTURE OF PLATE'''
| |
| | | | |
| | '''VI) Conclusion''' | | '''VI) Conclusion''' |
| - |
| |
| | | | |
| | |} | | |} |
- Small Phage September - October Notebook: Experiments
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|
- Small Phage
- March-April
- May-June
- July-August
- September-October
|
10.18 Cloning T7 Capsid Protein into iGEM Registry
I) Purpose
- Clone both WT and mutant T7 Capsid Protein into iGEM registry
II) Expected Outcome
- WT and mutant T7 capsid protein cloned into the iGEM registry.
III) Reagents Used
- Reagents required for cloning: dNTP, Phusion polymerase, butter, ddH2O, etc.
- Mutant phage S4, S10, S21, and L8; WT phage
IV) Procedure
1) Amplification and purification of insert (10.18)
- I. DNA purification
- * Put 5 ul of phage sample (S4, S10, S21, L8, and WT) and 45 ul of ddH20 into in PCR tubes with respective labeling.
- * Boil for 12 minutes in the PCR machine (98 Celsius).
- * Remove the tubes from the PCR machine and keep on ice until use in PCR.
- II. PCR w/ Phusion polymerase
- * To a eppendorf tube, add (for 6 PCR reactions simultaneously):
- - 210 uL of ddH2O
- - 60uL 5x Phusion buffer
- - 9uL 10mM dNTPs
- - 6uL of each primer (BI309 and BI310)
- * Mix well
- * To six new PCR tubes, labeled S4, S10, S21, L8, and WT respectively, add 2uL of template DNA from the boiled samples.
- * Add 3uL of Phusion Polymerase to the eppendorf tube (master mix).
- * Add 48uL of the master mix into each PCR tube.
- * Run 35 cycles with temperatures of 95 C, 50 C, and 72 C with an extension time of 1.5 minutes.
- * Leave overnight in the freezer.
- III. Low melt gel electrophoresis (10.19)
- * Add 75 ml of 1X TAE buffer and 0.75 grams of low melt agarose to an Erlenmeyer flask. Put into the microwave for about 90 seconds or until the agarose is completely dissolved. 1 drop of ethidium bromide was then added and mixed with the gel.
- * Pour the liquid onto the gel bed and let it cool. Remember to insert a sample comb.
- * The gel allowed to sit for overnight in the fridge to solidify.
- * Move the gel into the proper orientation (DNA runs towards the red electrode) in the gel box, cover the gel with 1X TAE buffer and load 8 ul of each of the PCR products with 3 ul of loading dye mixed in. Add a DNA ladder as a reference. Turn on the power supply and run at 80 volts.
- This is the procedure we used for our first round of electrophoresis. Unfortunately, the sample produced will not be enough for ligation (40-50uL of sample is need for loading). Thus we had to repeat this procedure.
V) Results
VI) Conclusion
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"
