Cidia+G.+(Vet)

= Fall 2014 =

Sonication (11/14/14)
The cells within the samples from 11/6/14 were lysed using a sonicator.The timed steps during sonication were not always accurately timed, and this may have had an effect on the result of the sonication. After sonication, the samples were stored within the -80 freezer.

Large Culture Step (11/6/14)
Large cultures were made using the small cultures made on 11/5/14. Two 4 liter flasks were filled with 1 liter of LB each and increments of the small cultures were added until the optical density reached 0.1 when read on a spectrophotometer. After reaching 0.1, the cultures were placed into an incubator to enable faster growth. Once the OD measured 0.5, the cultures were induced with IPTG and protein was made for 4 hours in the shaking incubator. Each culture was then spun down and the pellets of cells were then re-suspended in lysis buffer and stored in the -20 freezer for later sonication.

Small Culture Option B (11/5/14)
Small cultures were made using colonies from the BL21(DE3) FtHAP plates made on 10/16/14 for use in large scale expression and induction to produce FtHAP protein. Once the cultures were allowed to incubate for 16 hours, they exhibited colony growth which was the intended result. These small cultures will be used in large scale expression.

Making LB (11/5/14)
One liter of LB was made using 10 grams of bacto tryptone, 5 grams of NaCl and 10 grams of yeast extract. This solution was then autoclaved using the older autoclave liquid setting. Once autoclaving was done, it was observed that some of the LB agar may have boiled out of the flask that held it. This LB was made for use in large scale expression.

Secondary PCR (10/24/14)(11/5/14)
A secondary PCR was carried out in order to amplify the full length gene sequence for Ft AcpA for use in PCR Squared. This was done using the primary PCR sample from 10/7/14. On 11/5/14 a sample of this secondary PCR was ran on an agarose gel in order to view the result of the procedure. The result is seen below: Since no bands were seen in the gel when placed under UV light, the result of this procedure did not succeed, and another secondary PCR will have to be done in order to attain the full length AcpA sequence for later use in PCR Squared.

Making Plates (10/24/14)
LB Agar was made in the amount of 400 mLs for use in making plates of LB+Kan+Suc. The autoclave was down at the time so I was unable to autoclave the LB Agar in order to continue with the procedure. The LB Agar will be autoclaved at a later date in order to continue this procedure and produce 20 LB+Kan+Suc plates.

Expression of Ft HAP (10/21/14)
A small culture was made by placing 5 colonies from the plates of BL21(DE3) cells with Ft HAP within into 5 culture tubes containing 4 mL of LB and 4 uL of kan each. After allowing these cultures to grow up for approximately 10 hours, large scale expression of Ft HAP was attempted by adding the small culture in small amounts to a flask of LB with kanamycin in order to reach an optical density of 0.5 for induction with IPTG. After all small culture was added to the flask, the OD was only at 0.014 which was far too low for induction. This culture had to be disposed of. A new round of expression will be done using a larger small culture in order to successfully induce expression of Ft HAP protein with IPTG once the OD is 0.5.

Transformation of Ft HAP (BL21(DE3)) (10/16/14)
I decided to pick up on the already-cloned target, Ft HAP, belonging to my former TA Joey Olmos. Transformation of Ft HAP was done into BL21(DE3) cells in order to grow up colonies for use in making small cultures for large scale protein expression. After combining the Ft HAP DNA and the BL21-DE3 competent cells with SOC Media and kanamycin and allowing them to grow up briefly in culture tubes, the mixture was spread in 10 and 50 uL amounts onto two plates made with LB and Kanamycin. The plates after a 2 day incubation are seen below. Colonies from these plates will be used to make small cultures for use in large scale protein expression.

DNA Sequencing (10/16/14)
DNA Sequencing was done using samples 1 through 3 from the Miniprep done on 10/15/14 in order to determine if a positive Ft AcpA clone was achieved. After analyzing the results of nucleotide BLASTs done comparing the DNA sequencing results to that of the known sequence, it was determined that a positive clone was not achieved. Only the beginning and end of the sequence of Ft AcpA was present within the pNIC-Bsa4 vector. This could be due to the errors made in cohesive end generation. Another round of cloning will be done after doing another secondary pcr in order to amplify more full length Ft AcpA DNA for use in PCR Squared and either gel extraction or PCR Cleanup.

Miniprep (10/15/14)
Miniprep was carried out in order to isolate the DNA from the cells which were taken from the cultures made on 10/14/14. Three samples corresponding to the three cultures were made. The results after eluting the DNA and using Nanodrop to determine the concentrations are seen below. Sample 1: Concentration: 34.05 ng/uL Yield: 1,702.5 ng Sample 2: Concentration: 43.2 ng/uL Yield: 2,160 ng Sample 3: Concentration: 36.3 ng/uL Yield: 1,815 ng

Master Plate and Cultures (10/14/14)
A master plate and cultures were made using the colonies grown up during annealing and transformation done on 10/9/14. Three colonies were used to make three cultures which were incubated for approximately 16 hours and spun down. The supernatant was discarded and the pellets were kept which can be seen below. The master plate was allowed to grow for 3 days and is also seen below.

Gel Extraction (10/14/14)
Gel extraction was carried out using the two PCR squared gels made directly before. All 8 bands of DNA from both gels were extracted, but during the purification process no isopropyl was added before centrifuging. This was a large error results in termination of the procedure and disposal of the sample.

PCR Squared (10/14/14)
A PCR Squared was done using secondary PCR sample from 10/9/14 in order to attain bands of Ft AcpA DNA for use in Gel Extraction. All of each of the 8 samples were used and ran on two gels. Results are seen below.

Annealing and Transformation (10/9/14)
Annealing and transformation was carried out using the accepting vector and insert which were just made. Tubes with the following ratios were made, transformed, incubated, and spread onto separate LB+Kan+Suc plates for a 48 hour incubation. Tube A- 1:5 Tube B- 2:6 Tube C: 3:7 The plates after a 48 hour incubation are shown below. The colonies seen on Plate A (1:5) will be used to make a master plate and cultures in order to carry out miniprep and DNA sequencing. **Cohesive End Generation (10/9/14)** Cohesive end generation was carried out using the pNIC-Bsa4 cut on 10/8/14 as the accepting vector and the extracted DNA from 9/26/14 as the insert. Originally, twice the insert was supposed to have been made; however, only twice the DNA was added. I am also unsure if nanopure was added to either tube before allowing the cohesive ends to generate. Nonetheless, these samples will be used in annealing and transformation in order to attempt to positively clone Ft AcpA.

Diluting Primers (10/9/14)
The primers Ft_AcpA for and Ft_AcpA rev were diluted from 100mM to 20mM using nanopure for use in PCR Procedures.

PCR Squared (10/9/14)
A PCR Squared was carried out using freshly diluted primers and a sample of the secondary PCR done on 10/8/14. The results are seen below. The PCR squared samples are seen in lanes 5-8. Since there aren't strong bands in all 4 lanes, the master mix was most likely not mixed well enough. Since there is not a high enough amplification of the Ft AcpA sequence in all 4 samples, these samples will not be used in PCR Cleanup.

Secondary PCR (10/9/14)
A secondary PCR was carried out using freshly diluted primers in order to achieve a better result than the previous attempt (10/8). Results are seen below. The band in the 3rd lane indicates a successful secondary PCR. The band is of the correct size indicating the amplification of the Ft AcpA DNA sequence. A sample of this solution will be used to perform a PCR squared reaction.

Cut pNIC-Bsa4 (10/8/14)
pNIC-Bsa4 was digested with BsaI in order to remove the SacB gene in preparation for cloning. Results are seen below. Due to the two bands visible in lane 5 of the gel, the procedure can be considered a success as the band around 6.0 kbp is the cut pNIC-Bsa4, and the band around the 2.0 kbp band is the cut out SacB gene. A sample of the secondary PCR done on 10/8 was also ran on this gel in order to see if the result would differ. This gel shown no band for secondary PCR. The cut pNIC-Bsa4 will be used in cohesive end generation.

Secondary PCR (10/8/14)
A secondary PCR was carried out using 1 uL of the Primary PCR solution that was made yesterday in order to amplify the full Ft AcpA DNA sequence. Results of this procedure are seen below. Since a light band is seen around the 1.5 kbp DNA ladder marker, this procedure cannot be marked as a failure. A new secondary PCR will be carried out with fresh primers in order to achieve a better result. A PCR squared will also be carried out with a sample of this reaction in order to determine if this is a success. A round of cloning with the gel extracted on 9/26/14 and new cut pNIC will be carried out in order to make efficient use of my time.

Primary PCR (10/7/14)
A primary PCR was carried out using 1 uL of the new oligo mix that was just made. Results of this procedure are shown below. Since a smear was seen in lane 2, it was determined that this primary PCR was successful. A sample of this solution will be used as a template for a secondary PCR.

Making New Oigo Mix (10/7/14)
A new oligo mix was made using the 38 primers of my Ft AcpA sequence in order to being a new round of PCRs and cloning. There was no known error made during the making o the oligo mix.

DNA Sequencing (10/3/14)
DNA sequencing was carried out using the samples of DNA isolated during miniprep on 10/2/14. After analyzing the BLAST results of the known sequence of Ft AcpA against the results of DNA sequencing, it was determined that I had not achieved a positive clone. The BLASTs showed that only the beginning and end of the Ft AcpA sequence was being incorporated into the plasmid. Another round of cloning will be carried out in hopes of achieving a positive clone.

MiniPrep (10/2/14)
Miniprep was carried out using the spun down pellets of the 4 culture tubes made on 10/1/14. After the procedure was complete, the isolated DNA was nanodropped. Results are shown below. Sample 1. Average Concentration: 71.15 ng/ul, Yield: 3557.5 ng Sample should contain isolated DNA of Ft AcpA within pNIC-Bsa4. Sample 2. Average Concentration: 61.65 ng/ul, Yield: 3082.5 ng Sample should contain isolated DNA of Ft AcpA within pNIC-Bsa4. Sample 3. Average Concentration: 44.2 ng/ul, Yield: 2210 ng Sample should contain isolated DNA of Ft AcpA within pNIC-Bsa4. Sample 4. Average Concentration: 48.15 ng/ul, Yield: 2407.5 ng Sample should contain isolated DNA of Ft AcpA within pNIC-Bsa4.

These sample will be used in DNA sequencing in order to determine if I have achieved a positive clone.

Master Plate and Culture (10/1/14)
A master plate and cultures were made with the plates grown on 9/18/14. The cultures were allowed to incubate for 16 hours. The master plate was allowed to incubate for 3 days. Results are shown below. Since the cultures seen are cloudy, growth occurred during the incubation period. These cultures will be spun down and used in Miniprep in order to isolate the DNA within.

Gel Extraction (9/26/14)
Gel extraction was carried out by extracting the bands from lane 1 and 3 from gel 1 and from lanes 2 through 5 on gel 2. The DNA within the bands was then purified with a gel extraction kit. The nanodrop results are below. These results were confusing due to the peak at 230 nm and the slight bump at 260 nm. Ideally, the peak should be at 260 nm. Since the concentration seen is high, this extracted DNA will be used in cohesive end generation.

PCR Squared for Gel Extraction (9/26/14)
A PCR Squared reaction was carried out with sample 10/7 because sample 10/6 was left out from 9/23 to 9/26 and was, therefore, untrustworthy. Results of this reaction are shown below. Since bands were seen in lanes 1 and 3 of gel 1 and in lanes 2 through 5 of gel 2, gel extraction will be carried out using these bands.

Secondary PCR Gel (9/23/14)
A secondary PCR gel was done in order to determine if the sample was still good for use in the making of a PCR squared. Sample 10/6 was used. Results are below. ' From analyzing this gel, it was determined that the sample had not been degraded and could be used as a template in a PCR squared reaction. A PCR squared reaction will be carried out with either this sample or one from the same secondary PCR reaction in order to acquire bands of DNA for use in gel extraction.

PCR Squared for Gel Extraction (9/19/14)
PCR Squared was carried out in order to attain bands on DNA for use in gel extraction. Secondary sample 10/4 was used in order to make the PCR Squared. Gel results are seen below: Since no bands were seen on either gel, another PCR Squared will be done in order to acquire bands for use in gel extraction. A secondary PCR will be done first in order to determine if the samples from the secondary are still usable as templates for a PCR squared reaction.

Annealing & Transformation (9/18/14)
Annealing and transformation was carried out in order to insert the plasmid with the insert into DH5a cells in order to grow them up on plates. Plates were made with the following ratios of accepting vector to insert: A- 2:4 B- 2:5 C-2:3 D-3:4 After allowing the plates to incubate overnight: Plate A: One large colony seen as well as many sucrose crystals Plate B: One colony seen as well as many sucrose crystals Plate C: No colony growth seen. Many sucrose crystals seen. Plate D: Large colony seen. Many sucrose crystals seen. The colonies grown on these plates will be used to make a master plate and cultures in order to carry out miniprep and DNA sequencing.

**Cohesive End Generation (9/18/14)** Cohesive End Generation was carried out in order to create sticky ends on the accepting vector and the insert. The accepting vector was the pNIC-Bsa4 cut on 9/9/14. The insert was 12 ul of the extracted gel from column 1. When generating the cohesive ends, the tubes were placed in the 75 degree (Celsius) water bath for approximately one minute before it was taken out and left at room temperature for 45 minutes. The accepting vector and insert tubes were then allowed to heat inactivate for 20 minutes at 75 degrees Celsius. These samples will be used in annealing and transformation in order to hopefully attain a positive clone.

Making Sucrose & Agar Plates (9/15/14)
Plates were made for use in annealing and transformation. 500 mLs of 20% sucrose was made and 66.67 mls of the sucrose was added to 300 mls of LB Agar. Kanamycin was then added in the appropriate amount, and the mixture was pipetted onto plates in 20 mL increments.

DNA Sequencing (9/12/14)
DNA Sequencing was carried out using the isolated DNA in samples 1 through 8. After getting the results back, the BLASTs indicated that there was no positive control. Only the beginning and the end of the DNA sequence of Ft AcpA is being accepted into the plasmid. Another round of cloning will be done in order to achieve a positive clone.

Miniprep (9/10/14)
Miniprep was attempted in order to isolate the DNA within the cultures which were first spun down in order to obtain pellets. During the spin down steps, mot of the lysate came out of samples 5 and 1. Some came out of 8 and 4. There was also an error in eluting where sample 6 was eluted with 100 uL instead of 50 uL. Sample 1: Average Concentration: 60.2 ng/uL, Yield: 3,010 ng Sample should contain isolated DNA of Ft AcpA within pNIC-Bsa4. Sample 2: Average Concentration:106.8 ng/uL, Yield: 5,340 ng Sample should contain isolated DNA of Ft AcpA within pNIC-Bsa4. Sample 3: Average Concentration: 154.85 ng/uL, Yield: 7,742.5 ng Sample should contain isolated DNA of Ft AcpA within pNIC-Bsa4. Sample 4: Average Concentration: 150.15 ng/uL, Yield: 7,507.5 Sample should contain isolated DNA of Ft AcpA within pNIC-Bsa4. Sample 5: Average Concentration: 59.5 ng/uL, Yield: 2,975 ng Sample should contain isolated DNA of Ft AcpA within pNIC-Bsa4. Sample 6: Average Concentration: 46.15 ng/uL, Yield: 4,615 ng Sample should contain isolated DNA of Ft AcpA within pNIC-Bsa4. Sample 7: Average ConcentrationL 76.95 ng/uL, Yield: 3,847.5 ng Sample should contain isolated DNA of Ft AcpA within pNIC-Bsa4. Sample 8: Average Concentration: 84.9 ng/ul, Yield: 4,245 ng Sample should contain isolated DNA of Ft AcpA within pNIC-Bsa4.

These samples will be used in DNA sequencing in order to determine if I've made a positive clone.

New Cultures from Master Plate (9/9/14)
New cultures were made for colonies 1 through 8 using the master plate made on 9/8/14. The colonies are seen below after a 16 hour shaking overnight incubation period at 37 degrees Celsius. These cultures were very similar to those made on 9/8/14.

Mini-Prep (9/9/14)
Mini-Prep was attempted in order to isolate the DNA within the cultures which were first spun down in order to obtain pellets. In this procedure, a fatal error was made when step 3 was done before steps 1 and 2 causing all samples to be thrown away due to neutralization before re-suspension and lysis. A second set of cultures will be made in order to carry out a successful MiniPrep.

Making LB + Agar for use in LB+Kan+Suc Plates (9/9/14)
Originally, LB+Kan+Suc plates were supposed to be made today; however, we "ran out" of Sucrose, and I was unable to make the 20% sucrose necessary. Dr. B informed me that we did in fact have sucrose in a bucket, and the LB+Agar was made and autoclaved today in order to be used once 20% sucrose was obtained. 200 mL of LB+Agar was made, autoclaved, and stored in the 4 degree Celsius fridge.

RE Digest of pNIC-Bsa4 (9/9/14)
An RE Digest of pNIC-Bsa4 was carried out in order to prepare the accepting vector for use in cloning. The procedure was carried out according to direction, and a gel of sample after PCR-Cleanup, along with the PCR-Cleanup NanoDrop results, is seen below. Results of samples 1 and 2 of the PCR Cleaned RE Digested pNIC-Bsa4. The result labeled "sample 3" is really sample 1. The concentrations of both average to 110.85 ng/uL which indicates a yield of 3,325.5 ng of DNA within 30 uL. This concentration is high enough for use in cohesive end generation.

Master Plate and Cultures (9/8/14 - 9/9/14)
A master plate and cultures were made for 8 colonies from Plates A, B, and C as seen in Annealing and Transformation 9/5/14. The master plate and cultures protocol was followed normally. The results of this procedure are seen below. Master Plate made with 8 colonies from plates A through C. All colonies except for colony one grew well. This is consistent with the cultures which had the same result.

Cultures of colonies 1 - 8 seen from left to right after being allowed to shake in the incubator overnight at 37 degrees Celsius. All colonies except colony number 1 exhibit cloudiness and growth.

These cultures will be used in Mini-Prep in order to isolate the DNA within for use in DNA Sequencing.

Annealing and Transformation (9/5/14)
Annealing and Transformation was carried out and 3 Tubes of varying ratios of Accepting Vector to Insert within DH5alpha cells were made to be plated. __Tubes__ A - 2:4 B - 2:3 C - 4:4 The contents of each tube were emptied onto separate plates of LB+Kan+Suc labeled A, B, and C for incubation overnight at 37 degrees Celsius. Plates A, B, and C after incubation are shown below. Plate A 2:4 Colony growth is seen throughout in addition to many sucrose crystals. Plate B: 2:3 Colony growth is seen throughout in addition to many sucrose crystals. Plate C: 4:4 Colony Growth is seen throught in addition to many sucrose crystals.

8 Large Colonies from these plates will be taken and used in the making of a Master Plate and Cultures. The sucrose crystals should not have really had an effect on the colony growth. However, many tiny dots are seen throughout all of the plates, and I am unsure if this is contamination of some sort or crystals. Nonetheless, colonies will be used.

Cohesive End Generation (9/5/14)
Cohesive End Generation was carried out using the pNIC-Bsa4 which was cut yesterday as the Accepting Vector and the DNA purified from Gel Extraction as the Insert. Sticky ends were generated with the use of T4 DNA Polymerase and dCTP and dGTP in order to facilitate to incorporation of the Insert into the Accepting Vector.

RE Digest of pNIC-Bsa4 (9/4/14)
An RE-Digest of pNIC-Bsa4 with the enzyme BsaI-HF was carried out in order to cut out the Sac-B suicide gene in order to prepare the vector for the acceptance of the gene of interest (AcpA). Following RE-Digest, a sample of the cut pNIC-Bsa4 was ran on an agarose gel to ensure that pNIC had in fact been cut. Results are shown below. The band between the 6.0 and 5.0 kbp marks and the band slightly below the 1.5 kbp mark band indicate a successful RE-Digest of pNIC-BSa4. The band slightly below the 8.0 kbp mark may be uncut pNIC-Bsa4. This sample well be used in cohesive end generation due to the high concentration of cut pNIC-Bsa4.

Gel Extraction Cont. (9/2/14)
After retrieving the gel from the -20 Freezer, the second half of gel extraction was carried out and the DNA within the gel was purified in a similar fashion to PCR Cleanup with the addition of dissolving the gel in a gel solubilization solution. After the completion of this procedure, the eluted samples were nanodropped in order to determine the concentration and yield. Results are shown below

Average Concentration: 57.55 ng/uL Average Yield: 1438,75 ng

Average Concentration: 33.75 ng/uL Average Yield: 843 ng

Column 1 was shown to have a decently high yield, and this column will be used in cohesive end generation as the insert for the next round of cloning