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Team:Lethbridge/Notebook/Lab Work/Group2
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Sequencing data from samples sent to Genewiz come back showing k331025 and k331008, a signal sequence for localization to the outer membrane of ''E. coli'' is the correct sequence and confirmed suspicions regarding what was thought to be xylE in puc19. Two more attempts at restriction free cloning fail to produce viable colonies, including attempts to use non gel extracted PCR products that have had the original plasmid digested. K331008 is re-transformed, colonies picked, cultures grown, and a glycerol stock is made. <br> | Sequencing data from samples sent to Genewiz come back showing k331025 and k331008, a signal sequence for localization to the outer membrane of ''E. coli'' is the correct sequence and confirmed suspicions regarding what was thought to be xylE in puc19. Two more attempts at restriction free cloning fail to produce viable colonies, including attempts to use non gel extracted PCR products that have had the original plasmid digested. K331008 is re-transformed, colonies picked, cultures grown, and a glycerol stock is made. <br> | ||
==Week 12 July 18 - 24== | ==Week 12 July 18 - 24== | ||
| - | Another attempt at restriction free cloning with a different polymerase fails. Running low on PCR product mms6 and out of PCR product xylE. Strangely, an attempt at repeating the PCR with the same conditions fails to reproduce the xylE when run on an agarose gel. Another attempt at gel extracting mms6 PCR product and blunt end ligation with puc19 | + | ===Monday=== |
| + | Another attempt at restriction free cloning with a different polymerase fails. Running low on PCR product mms6 and out of PCR product xylE. | ||
| + | ===Tuesday, Wednesday, & Thursday=== | ||
| + | Strangely, an attempt at repeating the PCR with the same conditions fails to reproduce the xylE when run on an agarose gel. Another attempt at gel extracting mms6 PCR product and blunt end ligation with puc19 has poor transformation efficiency and no white colonies. Another assembly, restriction, ligation, and transformation, is done on the two remaining miscellaneous parts to get them into psb1c3. | ||
| + | ===Friday=== | ||
| + | Colonies picked and grown in LB culture. They are mini-prepped late this evening.<br> | ||
==Week 13 July 25 - 31== | ==Week 13 July 25 - 31== | ||
| - | After hearing about a system for cloning PCR products, an attempt at reproducing the PCR products using a polymerase possessing template independent terminal transferase activity that produces adenine overhangs is done but, | + | ===Monday=== |
| + | After hearing about a system for cloning PCR products, Promega pGEM-T PCR cloing system, an attempt at reproducing the PCR products using a polymerase possessing template independent terminal transferase activity that produces adenine overhangs is done but, there is no observed amplification. | ||
| + | ===Tuesday=== | ||
| + | A gel of the two reassembled miscellaneous parts is consistent with expected sizes. | ||
| + | ===Wednesday, Thursday, & Friday=== | ||
| + | Three more attempts at PCR using different conditions to replace the PCR products either with or without adenine overhangs fail to produce expected products. The PCR cloning system from Promega is ordered along with a new polymerase with terminal transferase activity.<br> | ||
==Week 14 August 1 - 7== | ==Week 14 August 1 - 7== | ||
| - | A test is done using different generic primers and two different polymerases on a confirmed plasmid. The polymerase that produces adenine overhangs successfully produces a band of the expected size. Using this polymerase, another PCR of xylE is attempted, the largest yet, with a slightly longer elongation time and a wide variety of annealing temperatures and magnesium concentrations. All twenty four unique reactions produced a band at the expected size of xylE. <br> | + | ===Monday & Tuesday=== |
| + | A test is done using different generic primers and two different polymerases on a confirmed plasmid. The polymerase that produces adenine overhangs successfully produces a band of the expected size. | ||
| + | ===Wednesday & Thursday=== | ||
| + | Using this polymerase, another PCR of xylE is attempted, the largest yet, with a slightly longer elongation time and a wide variety of annealing temperatures and magnesium concentrations. All twenty four unique reactions produced a band at the expected size of xylE. <br> | ||
==Week 15 August 8 - 14== | ==Week 15 August 8 - 14== | ||
| - | The xylE PCR products had the original host plasmids removed in a digest before they were cloned using the newly arrived pGEM-T vector blue/white colony cloning system from Promega. PCR producing adenine overhangs for mms6 were completed successfully before they were gel extracted and cloned using the new system. Colonies of both cloning were picked, grown in liquid media, and mini-prepped. Restricting these isolated plasmids and running on | + | ===Thursday=== |
| + | The xylE PCR products had the original host plasmids removed in a digest before they were cloned using the newly arrived pGEM-T vector blue/white colony cloning system from Promega. | ||
| + | ===Friday, Saturday, & Sunday=== | ||
| + | PCR producing adenine overhangs for mms6 were completed successfully before they were gel extracted and cloned using the new system. Colonies of both cloning were picked, grown in liquid media, and mini-prepped. Restricting these isolated plasmids and running on an agarose gel shows bands of expected sizes for both mms6 and xylE. The PCR products are now successfully cloned into plasmids<br> | ||
==Week 16 August 15 - 21== | ==Week 16 August 15 - 21== | ||
| - | An assembly to attach the arginine tag to xylE is done with red/white colony screening. White colonies are picked, grown in | + | ===Monday & Tuesday=== |
| + | An assembly to attach the arginine tag to xylE is done with red/white colony screening. White colonies are picked, and grown in LB culture. The two remaining miscellaneous samples are sent for sequencing along with the mms6 and xylE. | ||
| + | ===Wednesday=== | ||
| + | When restricted and run on a gel no colony for the xylE assembly has a band at the expected size. Sequencing data comes back. Sequencing data from Genewiz confirms k331022, a ribosomal bind site and yellow fluorescent protein with an n-terminally tagged arginine, and k331024, a ribosomal binding site and cyan fluorescent protein with an n-terminally tagged arginine. | ||
| + | ===Thursday=== | ||
| + | Further analysis of sequence data confirms the successful PCR and cloning for mms6 and xylE. <br> | ||
==Week 17 August 22 - 28== | ==Week 17 August 22 - 28== | ||
| + | ===Monday, Tuesday, Wednesday, Thursday, & Friday=== | ||
Ryan is on vacation on Vancouver island. The arginine tag is assembled onto xylE. <br> | Ryan is on vacation on Vancouver island. The arginine tag is assembled onto xylE. <br> | ||
==Week 18 August 29 - September 4== | ==Week 18 August 29 - September 4== | ||
Revision as of 14:22, 28 September 2011
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