Protocols
- Blunt end ligation is the same protocol as standard ligation except 2 hour minimum room temperature incubation time.
- Restriction free cloning is the same protocol as a standard PCR except PCR products from a different PCR reaction are used in place of conventional primer oligos. This is done to "insert" the PCR product into the template DNA.
Week 3 May 16 - 22 2011
Ryan vacationing in Whitefish. Having an awesome time.
Week 4 May 23 - 29 2011
Monday
First week Ryan begins working full time in the lab.
Tuesday
Successfully used polymerase chain reaction (PCR) and specifically designed primers to alter prefix and suffix regions to to the Silver standard for the genes xylE and mms6. This is done to allow the fusion of polypeptide sequences to both genes. On an agarose gel the DNA strands are of expected size for both xylE and mms6 with the fusion standard.
Wednesday, Thursday, & Friday
After attempting to cut PCR products of xylE and mms6 and cloning into psb1c3 using red/white screening, no white colonies test positive for the product when the plasmids are restricted and resolved on a gel. Miscellaneous plasmids are taken out of the glycerol stock and grown in LB cultures and mini-prepped the next day.
Week 5 May 30 - June 5 2011
Monday
The problem with the ligation of PCR products into psb1c3 is diagnosed to be the result of prefix and suffix restriction sites being too close to the ends of the PCR products.
Tuesday & Wednesday
Instead of using red/white colony screening and psb1c3 the polymerase used in the PCR produces blunt ends and therefore a blunt end ligation is used for cloning with puc19 instead of the conventional ligation. Different conditions are used in the blunt end ligation. Since puc19 has to be cut bluntly half the samples have the blunt end restriction enzyme heat killed after the restriction. Blue/white colony screening has to be used for puc19 cloning which requires X-Gal and IPTG. Solutions of these are made for the plates. Attempt to restrict, ligate, and transform in an assembly miscellaneous parts from synthesis vectors to psb1c3.
Thursday & Friday
Results from growing colonies in overnight LB cultures, mini-prepping, restricting, and running them on an agarose gel confirm a white colony for k331025, an enhanced cyan fluorescent protein with a ribosomal binding site and a c-terminally fused arginine residue tail. One colony for the blunt end ligation cloning of an xylE PCR product produces a band at the expected size when the plasmid is restricted and run on a gel. Attempts to move the remaining miscellaneous parts into psb1c3 produce no positive testing white colonies.
Week 6 June 6 - 12
Monday & Tuesday
A glycerol stock of the one colony of xylE and k331025 were made after they were re-transformed and grown in liquid LB culture.
Wednesday, Thursday, & Friday
After another attempt the mms6 PCR product still refuses to be cloned via blunt end ligation. The same can be said for the another conventional assembly of the miscellaneous parts to psb1c3.
Week 7 June 13 - 19
Monday
A plasmid preparation of S04261, the ten residue arginine tag with a double transcriptional terminator, is transformed. A blunt end ligation between mms6 and puc19 is left over night.
Tuesday
A colony of S04261 is picked and grown in LB media. The blunt end ligation of mms6 is transformed. An assembly of the miscellaneous parts where double restriction solution is used for the ligation is carried out to the transformation.
Wednesday
A glycerol stock is made from S04261. Whitish colonies are picked from the mms6-puc19 plates and grown in LB cultures overnight.
Thursday
Mini-prep of colonies picked from mms6-puc19 plates. Restriction and agarose gel of the extracted plasmids fails to confirm successful cloning. White colonies are picked from the miscellaneous parts plates and grown in LB culture overnight.
Friday
Mini-prep of colonies picked from miscellaneous parts plate. Restriction and agarose gel of exteacted plasmids fails to confirm successful cloning.
Week 8 June 20 - 26
Monday
The xylE from puc19 is restricted, ligated, and transformed into psb1c3 for red/white colony screening with the attachment of S04261, the arginine tag and double terminator. Diminished supplies of various mini-prepped plasmids picked from glycerol stocks and are grown in LB cultures overnight
Tuesday
Colonies are picked and grown in LB culture overnight. Cultures of various plasmids are mini-prepped.
Wednesday, Thursday, & Friday
The miscellaneous parts are re-assembled into psb1c3. Both through PCR and restriction the gels do not show a positive result for the xylE-arg tag assembly. A blunt end ligation of mms6 into puc19 shows some white colonies.
Saturday & Sunday
Colonies of the miscellaneous plasmids and mms6 are picked and grown in LB culture. They are then mini-prepped.
Week 9 June 27 - July 3
Monday
The miscellaneous parts are restricted and run on a gel along with mini-preps of the mms6 colonies and, while all the remaining miscellaneous parts are consistent with expected results the mms6 is not observed on the gel.
Tuesday
A PCR of the mms6 in puc19 also gives the same result on an agarose gel as the restriction. The xylE is restricted, ligated, and transformed into psb1c3 while attaching S04261, the arinine tag with double terminator.
Wednesday
Re-assembled, plated, possible colonies are picked and grown in liquid LB media overnight. PCR of miscellaneous parts when run on a gel are consistent with expected results.
Thursday & Friday
Overnight cultures are mini-prepped and both PCR and restriction are done yet when run on an agarose gel the results are negative for a successful assembly.
Week 10 July 4 - 10
Monday, Tuesday, & Wednesday
The lack of progress on xylE and mms6 being suspicious, new approachs are used. PCR products of both xylE and mms6 with the fusion standard are gel extracted using a Qiagen gel extraction kit. These gel extractions have DNA presence confirmed on a gel and are then used in a blunt end ligation with puc19.
Thursday & Friday
An initial attempt at restriction free cloning with psb1c3 is made. Neither approach produces viable colonies.
Week 11 July 11 - 17
Monday & Tuesday
Samples are prepared and sent for sequencing. Another attempt at restriction free cloning is made using gel extracted PCR products but does not produce viable colonies.
Wednesday, Thursday, & Friday
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.
Week 12 July 18 - 24
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.
Week 13 July 25 - 31
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.
Week 14 August 1 - 7
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.
Week 15 August 8 - 14
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
Week 16 August 15 - 21
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.
Friday
Primers are ordered for altering other genes in the xylene degradation pathway besides xylE.
Week 17 August 22 - 28
Monday, Tuesday, Wednesday, Thursday, & Friday
Ryan is on vacation on Vancouver island. The arginine tag is assembled onto xylE.
Week 18 August 29 - September 4
Monday
The primers arrive to either add or alter the Silver fusion standard respective to more xylene degradation genes, xylT and xylF. Initial PCR produces expected bands when run on a gel, even for xylT which was in the genomic DNA given to us from David Houston.
Tuesday & Wednesday
Using pGEM-T both genes are cloned, however, the antibiotic on the plates are bad and we get a lawn of colonies since there is no selection pressure.
Thursday & Friday
Making, autoclaving LB agar to make new plates. The autoclave is being finicky and the LB agar is not sterilized until late Friday.
Week 19 September 5 - 11
Monday
Labour day break for group 2. Last day before we return to regular classes.
Tuesday & Wednesday
New plates are poured with the sterilized LB agar the two genes from the xylene degradation pathway are re-cloned with pGEM-T.
Thursday
Colonies are picked and grown in LB culture.
Friday
Cultures are mini-prepped. Plasmids are restricted and an agarose gel confirms the presence of both xylT and xylF in their respective plasmids.
Week 20 September 12 - 18
Monday
The genes for xylT and xylF were sent for sequencing.
Wednesday, Thursday & Friday
Sequence data gets back but the analysis is grim. The primers that were ordered were not designed properly and the stop codon in both sequences was not removed, defeating the purpose of having the fusion standard.
Week 21 September 19 - 25
Monday, Tuesday, Wednesday, Thursday, & Friday
Working on the presentation for aGEM conference.
Final Half Week September 26 - 28
Monday, Tuesday, & Wednesday
Working non-stop writing this notebook here in the wiki.
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