Research+Page-+Larry

Larry's Research Summer 2012 Week of 7/16/12 -In order to verify the primer overlap assembly, we will try to send to sequencing -Last week some of the PCR squared used for the last transformation was sent to sequencing, but results were crappy -Really poor coverage, however within that coverage is highly conserved matches -This could mean that the primers are failing to anneal correctly when sequencing -It could also mean that the oligonucleotides primers are not forming the correct gene. However, this is unlikely since the gel check of PCR squared indicates the correctly sized gene

-Want to perform PCR squared, but ran out of 2ndary PCR, so made more Lane 1: skip Lane 2: 1kb Ladder Lane 3: Andrew's primary PCR Lane 4: Andrew's secondary PCR Lane 5-6: Larry's secondary PCRs -I ran double the amount of PCR squared since I ran out of the last supply of 2ndary PCR quickly -Both samples look like they worked

-Ran PCR squared in preparation for gel extraction, so 8 50ul samples were made Lane 1: skip Lane 2: 1kb Ladder Lane 3-10: PCR squared -PCR squared looks successful, will proceed with gel extraction

-Performed gel extraction -Had 1.86g of gel -High concentration of GAPDH -Good 260/280 and 260/230 ratios indicates low contamination -Will use this to clone

-Cut pNIC-Bsa4 Lane 1: Skip Lane 2: 1 kb Ladder Lane 3: RE Digest of pNIC-Bsa4 with BsaI (AB) Lane 4: RE Digest of pNIC-Bsa4 with BsaI (YH) Lane 5: Skip Lane 6: PCR Clean Up (KR) Lane 7: PCR Clean Up (KR) Lane 8: RE Digest of pNIC-Bsa4 with BsaI (KR) Lane 9: pFAL (KR) -The 4th lane indicates a successful pNIC-Bsa4 cut

Week of 7/9/12 -Results of sequencing are in code NNNNNNNTTNACTTTAGNNGAGANNTACATATGCACCATCATCATCATCATTCTTCTGGTGTAGATCTGGGTACCGAGAA CCTGTACTTCCAATCCATGGCGGTTCGTGAACTGCCGGGTGCGTGGAACTTCCGCGACGTGGCTGACACCGCGACCGCGC TGCGTCCGGGTCGTCTGTTCCGTAGCTCTGAACTGTCTCGTCTCGATGATGCCGGTCGTGCCACCCTGCGCCGTCTGGGT ATCACCGACGTTGCCGATCTCCGCTCTTCCCGTGAAGTTGCTCGTCGTGGTCCAGGTCGTGTTCCAGATGGTATCGACGT TCACCTGCTGCCGTTTCCTGATCTCGCGGACGATGACGCGGATGACTCTGCGCCTCACGAAACCGCATTCAAACGTCTGC TGACCAACGACGGCTCTAATGGCGAATCTGGTGAATCTTCTCAGTCTATCAATGATGCAGCCACTCGTTACATGACCGAC GAATATCGTCAATTCCCGACCCGTAATGGTGCGCAGCGTGCCCTGCATCGTGTTGTAACCCTCCTGGCCGCTGGCCGTCC GGTTCTGACCCACTGCTTTGCGGGTAAAGACCGTACCGGCTTCGTTGTTGCACTGGTTCTGGAAGCGGTTGGTCTGGATC GTGACGTAATTGTGGCGGACTACCTCCGTTCTAACGACAGCGTTCCGCAACTGCGTGCGCGTATCTCTGAAATGATCCAG CAGCGTTTCGACACCGAACTGGCACCGGAGGTTGTTACCTTCACCAAAGCGCGTCTGTCTGACGGCGTTCTCGGTGTTCG TGCGGAATACCTGGCAGCGGCTCGTCAAACCATCGACGAAACTTACGGNTCTCTGGGTGGTTACCTGCGTGATGCGGGCA TCTCTCNNGCNACCGTTAACCGTATGCGTGGTGNTCTCCTGGGTTAACAGTAAAGGNGGATANGNATCCGAATTCGAGCT CCGTCGACAAGCTTGCGGCNCACTCNAGCACCACACCACCNCCACTGAGATCCGGCTGCTAACAAGCCCGAANGNAGCTG ANTTGGNTGCTGCACNNTGANCAANANTAGNATANNCNTNGGGNNNNNACGGGTCTGAGGGNTTTTNNTNANNNNNGANN NNNNNCCGNATGGNNANGGGNNNNNCNNNNNNNNNNTNANNNNNNNNNNNNNNNNNNNNNNNANNNGNNCNNNNNNNNNN NNNNNNNANNNNNN code -All 8 results are similar to this -When blasted, there is no similarity to my gene, or to any gene in the complete database -Did a blast on my entire virtual plasmid -The sequence shares ends with the virtual plasmid, so it means that something did get transformed -However, the stuff inserted is junk -This may be an issue with purity -Will attempt gel extraction to improve purity

Gel extraction: -Forgot to take a picture of the gel, but PCR squared was successful -Have the concentration -With a concentration as low as 37ng/ul, this sample won't be useful since PCR cleanup will remove too much DNA -The 260/230 ratio of .09 indicates much contamination, so we can't just use this for transformation

Week of 7/2/12 -Attempted transformation into DH5alpha again -Transformation looks successful, each of these colonies must have the sacB gene disrupted -Hopefully tbGAPDH is what disrupted sacB -Colonies lookin good =D

-Selected 8 of the colonies for masterplate -Grew up and did miniprep -Found concentration of all 8 samples -There are 7 other samples, but the concentration is similar. -Graphs suggest there is DNA, but we don't know for sure if it's the correct gene

-Sent the plasmid to sequencing -While waiting, I did an RE digest using BsaI, but it failed

Week of 6/25 -- Looks good Larry. -- Dr. B -Cut pNIC-Bsa4 as the first step in cloning -Very low concentration after the cut, so it looks like it failed.

-Reran the cut -However, ran out of material to Nanodrop since I had to use all of the material for the cohesive end generation (since I messed up pipetting) -Did run a gel beforehand Lane 1: skip Lane 2: 1kb ladder Lane 3: Andrew B's cut Lane 4: Larry's cut of pNIC-Bsa4 using BsaI -The cut looks successful since there are 2 major bands -The large band at the top of lane 4 is probably uncut plasmid

-Proceded to cohesive end generation/ligation, but I failed to grow up any colonies, so will restart cloning. -Performed another cut -Looks to be much more successful than the 1st attempt

Week of 6/18 -Did a test of 6 KOD hotstart polymerases using pNIC-Bsa4 and pLIC primers. -Used [|this] protocol Lane 1: skip Lane 2: 1kb Ladder Lane 3-8: KOD hotstart polymerase A-F Lane 9: Neg. control -Negative control did not work -Bands are at about 2kb, which is nearly the correct size pNIC-Bsa4 -**Tube E did not work, so don't use that one!**

Did primary and secondary PCR -Used sticky ends with the custom primers Lane 1: skip Lane 2: 1kb ladder Lane 3: primary pcr Lane 4: secondary PCR -Very bright band at about 1kb confirms the success of the primer overlap assembly.

Nanodropped the 2 PCR cleanup samples -Blanked using 10mM Tris ph 8.0 -Concentrations of tbGAPDH gene is a little low -May be difficult to get the gene cloned

Ran PCR squared through an agarose gel Lane 1: skip Lane 2: 1kb Ladder Lane 3: PCR Squared -the PCR squared seems to be at the 1kb mark, so it looks successful.

Week 14 Varying amounts of the enzyme YopH assayed with pNPP plotted against absorbance. Dark blue point indicates the optimal concentration for vary substrate assay. YopH assayed with varying amounts of substrate pNPP plotted against absorbance.
 * Instead of trying to continue expression (since this is the last week), I did a two assays for yoph.
 * When doing the enzyme concentration assay, I used a very high working enzyme concentration, but the assay still worked
 * The substrate assayed failed, since there is supposed to be a curve upwards, then a plateau.

Week 13 (Thanksgiving week)
 * Tried to grow up more BL21 for expression, but using an altered procedure.
 * Instead of growing an initial amount in 50ml of LB then transferring them the next day for spectrophotometry, the spectrophotometry would be done on the 50ml and after the OD was 0.3, IPTG would be added and then transferred to a large 2L flask for overnight growth
 * However, the OD600 would not go up even after measuring for a few hours
 * Tried to grow the BL21 using the normal procedure, but once again the OD600 would not increase. However this may be because I added the kanamycin 20 minutes after it I put it in the incubator.

Week 12 Cloudy solution indicates protein precipitate. mmmmm creamy...
 * Started purification
 * During the pH adjustment step, I used 5M NaOH and completely overshot the target pH. Then I tried to use concentrated HCl to counteract the NaOH, but this caused the protein to precipitate, making it unusable
 * Started a shortened version of expression, where the initial 50ml growth is used the same day as the 500ml growth.
 * Grew up the bacteria in 50ml until OD600 was around 1.0

Week 11
 * Started Expression
 * Pellet weighed 2.5 grams
 * Time || OD600 ||
 * 1:27 || .093 ||
 * 1:55 || .063 ||
 * 2:35 || .121 ||
 * 3:00 || .191 ||
 * 3:27 || .197 ||
 * 3:56 || .267 ||
 * 4:18 || .28 ||
 * The OD600 at 3:27 must have been an error in measurement
 * Stopped at .28 absorbance since it looked like the growth was starting to plateau

Week 10
 * Tried to do transformation again but no colonies grew on the master plate
 * Decided to use Justin's BL21 colonies and proceed to expression

Week 9
 * Only one colony had any results, but there were deletions[[image:Blast_Identity_Sample_1.png width="800" height="303"]]
 * Started 2nd runs of HF9 and MW libraries

Week 8 Nanodrop of the three colonies that grew.
 * Selected colonies onto a master plate
 * Grew up colonies and performed miniprep
 * Only three colonies grew
 * Also began virtual screening of PP2Ctg
 * Queued HF9 and MW libraries

Week 7 Plates of Kan+Sucrose. Plate A (left) has 4 ul of treated insert and Plate B (right) has 8 ul of treated insert.
 * Inserted PP2Ctg into pNIC-bsa4
 * Transformed plasmid into the bacteria

Week 6 PCR squared looks successful The yield from these four combined PCR squared is very high
 * Performed primary, secondary PCR, and PCR squared

Week 5 Lane 1: skip Lane 2: 100bp Ladder Lane 3-4: PCR squared
 * Performed PCR Squared
 * Performed a gel check (since primary/secondary was left out overnight...)
 * Found that everything broke down

Week 4 Going from right to left: Lane 1: Skip Lane 2: 100bp Ladder Lane 3: Gel extracted PCR squared Lane 4: 1kb Ladder Lane 5: Preparation of pNIC-Bsa4 for cloning Nanodrop of the gel extraction. There is no peak at 260, suggesting that there is no DNA left. Lane 1: skip Lane 2: 100bp ladder Lane 3: Primary PCR Lane 4: Secondary PCR
 * Did a gel check on the prep of pNIC-Bsa4 as accepting vector and the Gel extraction of the contaminated PCR squared
 * The band from the gel extraction is extremely faint, so the gel extraction process probably lost most of the DNA
 * A nanodrop reading confirms that there is nothing of use from the extraction
 * Completed primary and secondary PCR
 * The secondary PCR looks successful

Week 3 Lane 1: Skip Lane 2: 100bp Ladder Lane 3-6: PCR squared amplified
 * Ran PCR Squared on the summer PCR squared to amplify in preparation of gel extraction
 * Performed gel extraction on the top band

Week 2
 * Ran PCR Squared on the summer PCR squared to amplify in preparation of gel extraction
 * Accidentally froze gel, rendering it unusable

Week 1 Lane 1: Skip Lane 2: 100bp Ladder Lane 3: Skip Lane 4: PCR Squared from summer 2011
 * Ran a gel check on my PCR squared.
 * Found contamination

Lane 1: skip Lane 2: 1 kb DNA ladder Lane 3: Uncut plasmid Lane 4: EcoRI Lane 5: PvuII Lane 6: EcoRI+PvuII

Caption?? - is this deja vu PCR? 