Sarah+H.+(RP+Summ+14)

=WE LOVE VDS AND WILL MISS YALL. :)= =Week 9=

Day 28 (7/28/14) ...Day 28 just happens to be the 28th of July...you have just entered the twilight zone: We began making PFU polymerase by making an overnight culture. We also made a page gel and stored it in the 4C fridge. We worked on endnote. Super fun. We...meaning me...analyzed the DNA sequencing results for the PFU polymerase. These are the results for the PFU reverse sample. CNTTCGGGCTTTGTTAGCAGCCGGATCCTCGAGCTAGGATTTTTTAATGTTAAGCCAGGAAGTTAGGCCGACTTGTCTTGTCTTTTGGTATCTGAGGTCTTCCTTTCTGTATCCAAATCCCTCCAATATCCTAAGTACCGCTGGAAGAACCTGGTTCTCAATGTAATATTCTGCGTCATACTTGTGCTTTTTGGGATCGTATTCCTCAGCTAGAATTGCCCTATTGCTAATTGGACCATCGCCTCTAAGTACTATGTATCCAATTACCATTCCTGGCTTTATTTTAACTCCTTTAGCAGCTAGTTTCTTTGCAGCAGCTACGTGAGGACCTATCGCCTTATACTCATGTAATGGTCTTGTTATCTGCTCATATATTGCGAGCTTCTCTGGTGGAATTTCATAATTGGCAAGCTTTTGTATTACTTCTTTTACTATTCTCACAGCTTCTTCAACATCTCCGTGTTTTAGTATTGTCTCCAAAACTCTAGCTTGAGTTTCTTTTGCAATTTCACTCCAATCTCTCCTAACTATCTCTAAACCACGAGTAATGACTTTTCCTTCTTCATCTATTACTGCATACCTCTTCTTCGTAACGAAGAATCCCCTCTTATAAAACCCTTCATATTCAAGCTCTAGCAGTCCAGGGAGCTTTGAATTTATGTATTTTACAAATTCTAGAGCCTTTATCTTTATTTCCTCACTTTCTCCTCCTGGGNTAGTTGCATAGAGACCATCAGTGTCAATGTAGAGGACTTTAAATCCAAGCTTTTCTTCGAGCTCCTTCCATACTAACTCGATGTACTTTCTTCCCCNNNAGTAACGCTCTCAGCACACTCCTTACAGTACCATCTTGCTTTTGCATAGCCATAATATCCGTAGAAAGAATTTGCTAANAGTTTTATCGCTTTTTNNCNATAGTCANGNAGNATTTTTNCNATNNNNTCTNNNNTTCNNNCATNTTTNNNNNA
 * __ Revised Version with ‘N’s Cut Out __**

Reverse Complement results >Untitled reverse complement TNNNNNAAANATGNNNGAANNNNAGANNNNATNGNAAAAATNCTNCNTGACTATNGNNAA AAAGCGATAAAACTNTTAGCAAATTCTTTCTACGGATATTATGGCTATGCAAAAGCAAGA TGGTACTGTAAGGAGTGTGCTGAGAGCGTTACTNNNGGGGAAGAAAGTACATCGAGTTAG TATGGAAGGAGCTCGAAGAAAAGCTTGGATTTAAAGTCCTCTACATTGACACTGATGGTC TCTATGCAACTANCCCAGGAGGAGAAAGTGAGGAAATAAAGATAAAGGCTCTAGAATTTG TAAAATACATAAATTCAAAGCTCCCTGGACTGCTAGAGCTTGAATATGAAGGGTTTTATA AGAGGGGATTCTTCGTTACGAAGAAGAGGTATGCAGTAATAGATGAAGAAGGAAAAGTCA TTACTCGTGGTTTAGAGATAGTTAGGAGAGATTGGAGTGAAATTGCAAAAGAAACTCAAG CTAGAGTTTTGGAGACAATACTAAAACACGGAGATGTTGAAGAAGCTGTGAGAATAGTAA AAGAAGTAATACAAAAGCTTGCCAATTATGAAATTCCACCAGAGAAGCTCGCAATATATG AGCAGATAACAAGACCATTACATGAGTATAAGGCGATAGGTCCTCACGTAGCTGCTGCAA AGAAACTAGCTGCTAAAGGAGTTAAAATAAAGCCAGGAATGGTAATTGGATACATAGTAC TTAGAGGCGATGGTCCAATTAGCAATAGGGCAATTCTAGCTGAGGAATACGATCCCAAAA AGCACAAGTATGACGCAGAATATTACATTGAGAACCAGGTTCTTCCAGCGGTACTTAGGA TATTGGAGGGATTTGGATACAGAAAGGAAGACCTCAGATACCAAAAGACAAGACAAGTCG GCCTAACTTCCTGGCTTAACATTAAAAAATCCTAGCTCGAGGATCCGGCTGCTAACAAAG CCCGAANG

Translate results >rf 1 Untitled reverse complement XXKXXXXXXXXXKXLXDYXXKAIKLLANSFYGYYGYAKARWYCKECAESVTXGEESTSS* YGRSSKKSLDLKSSTLTLMVSMQLXQEEKVRK*R*RL*NL*NT*IQSSLDC*SLNMKGFI RGDSSLRRRGMQ**MKKEKSLLVV*R*LGEIGVKLQKKLKLEFWRQY*NTEMLKKL*E** KK*YKSLPIMKFHQRSSQYMSR*QDHYMSIRR*VLT*LLQRN*LLKELK*SQEW*LDT*Y LEAMVQLAIGQF*LRNTIPKSTSMTQNITLRTRFFQRYLGYWRDLDTERKTSDTKRQDKS A*LPGLTLKNPSSRIRLLTKPE

Translate results >rf 2 Untitled reverse complement XXXMXEXXXXXXKXXXTXXKKR*NX*QILSTDIMAMQKQDGTVRSVLRALLXGKKVHRVS MEGARRKAWI*SPLH*H*WSLCNXPRRRK*GNKDKGSRICKIHKFKAPWTARA*I*RVL* EGILRYEEEVCSNR*RRKSHYSWFRDS*ERLE*NCKRNSS*SFGDNTKTRRC*RSCENSK RSNTKACQL*NSTREARNI*ADNKTIT*V*GDRSSRSCCKETSC*RS*NKARNGNWIHST *RRWSN*Q*GNSS*GIRSQKAQV*RRILH*EPGSSSGT*DIGGIWIQKGRPQIPKDKTSR PNFLA*H*KILARGSGC*QSPX

Translate results >rf 3 Untitled reverse complement XXXXXXXRXXXKNXX*LXXKSDKTXSKFFLRILWLCKSKMVL*GVC*ERYXXGRKYIELV WKELEEKLGFKVLYIDTDGLYATXPGGESEEIKIKALEFVKYINSKLPGLLELEYEGFYK RGFFVTKKRYAVIDEEGKVITRGLEIVRRDWSEIAKETQARVLETILKHGDVEEAVRIVK EVIQKLANYEIPPEKLAIYEQITRPLHEYKAIGPHVAAAKKLAAKGVKIKPGMVIGYIVL RGDGPISNRAILAEEYDPKKHKYDAEYYIENQVLPAVLRILEGFGYRKEDLRYQKTRQVG LTSWLNIKKS*LEDPAANKARX

DNA polymerase [Pyrococcus furiosus] Sequence ID: ref|WP_011011325.1| Length: 775Number of Matches: 1 [|See 9 more title(s)]

Query 6 GRKYIELVWKELEEKLGFKVLYIDTDGLYATXPGGESEEIKIKALEFVKYINSKLPGLLE 65 GRKYIELVWKELEEK GFKVLYIDTDGLYAT PGGESEEIK KALEFVKYINSKLPGLLE Sbjct 518 GRKYIELVWKELEEKFGFKVLYIDTDGLYATIPGGESEEIKKKALEFVKYINSKLPGLLE 577
 * Alignment statistics for match #1 ||
 * **Score ** || **Expect ** || **Method ** || **Identities ** || **Positives ** || **Gaps ** ||
 * 501 bits(1291) || 3e-170 || <span style="font-family: Verdana,sans-serif; font-size: 9pt;">Compositional matrix adjust. || <span style="font-family: Verdana,sans-serif; font-size: 9pt;">254/258(98%) || <span style="font-family: Verdana,sans-serif; font-size: 9pt;">254/258(98%) || <span style="font-family: Verdana,sans-serif; font-size: 9pt;">0/258(0%) ||

Query 66 LEYEGFYKRGFFVTKKRYAVIDEEGKVITRGLEIVRRDWSEIAKETQARVLETILKHGDV 125 LEYEGFYKRGFFVTKKRYAVIDEEGKVITRGLEIVRRDWSEIAKETQARVLETILKHGDV Sbjct 578 LEYEGFYKRGFFVTKKRYAVIDEEGKVITRGLEIVRRDWSEIAKETQARVLETILKHGDV 637

Query 126 EEAVRIVKEVIQKLANYEIPPEKLAIYEQITRPLHEYKAIGPHVAAAKKLAAKGVKIKPG 185 EEAVRIVKEVIQKLANYEIPPEKLAIYEQITRPLHEYKAIGPHVA AKKLAAKGVKIKPG Sbjct 638 EEAVRIVKEVIQKLANYEIPPEKLAIYEQITRPLHEYKAIGPHVAVAKKLAAKGVKIKPG 697

Query 186 MVIGYIVLRGDGPISNRAILAEEYDPKKHKYDAEYYIENQVLPAVLRILEGFGYRKEDLR 245 MVIGYIVLRGDGPISNRAILAEEYDPKKHKYDAEYYIENQVLPAVLRILEGFGYRKEDLR Sbjct 698 MVIGYIVLRGDGPISNRAILAEEYDPKKHKYDAEYYIENQVLPAVLRILEGFGYRKEDLR 757

Query 246 YQKTRQVGLTSWLNIKKS 263 YQKTRQVGLTSWLNIKKS Sbjct 758 YQKTRQVGLTSWLNIKKS 775

These are the results for the PFU forward samples: ATTTTGTTTAACTTTAAGAAGGAGATATACCATGGGCCATCATCATCATCATCATCATCATCATCACAGCAGCGGCCATATCGAAGGTCGTCATATGATTTTAGATGTGGATTACATAACTGAAGAAGGAAAACCTGTTATTAGGCTATTCAAAAAAGAGAACGGAAAATTTAAGATAGAGCATGATAGAACTTTTAGACCATACATTTACGCTCTTCTCAGGGATGATTCAAAGATTGAAGAAGTTAAGAAAATAACGGGGGAAAGGCATGGAAAGATTGTGAGAATTGTTGATGTAGAGAAGGTTGAGAAAAAGTTTCTCGGCAAGCCTATTACCGTGTGGAAACTTTATTTGGAACATCCCCAAGATGTTCCCACTATTAGAGAAAAAGTTAGAGAACATCCAGCAGTTGTGGACATCTTCGAATACGATATTCCATTTGCAAAGAGATACCTCATCGACAAAGGCCTAATACCAATGGAGGGGGAAGAAGAGCTAAAGATTCTTGCCTTCGATATAGAAACCCTCTATCACGAAGGAGAAGAGTTTGGAAAAGGCCCAATTATAATGATTAGTTATGCAGATGAAAATGAAGCAAAGGTGATTACTTGGAAAAACATAGATCTTCCATACGTTGAGGTTGTATCAAGCGAGAGAGAGATGATAAAGAGATTTCTCAGGATTATCAGGGAGAAGGATCCTGACATTATAGTTACTTATAATGGAGACTCATTCGACTTCCCACATTTAGCGAAAAGGGCAGAAAAACTTGGGATTAAATTAACCATTGGAAGAGATGGAAGCGAGCCCAAGATGCAGAGAATAGGCGATATGACGGCTGTAGAAGTCANGGGAAGAATACATTTCGACTTGTATCATGTAATAACAAGGACAATAAATCTCCCAANATACACACTAGAGGCTGTATATGAANCAATTTTTGGAAAGCCNA
 * __ Revised Version with ‘N’s Cut Off __**

<span style="font-family: Arial,sans-serif; font-size: 10pt;">Translate results >rf 1 Untitled ILFNFKKEIYHGPSSSSSSSSSQQRPYRRSSYDFRCGLHN*RRKTCY*AIQKRERKI*DR A**NF*TIHLRSSQG*FKD*RS*ENNGGKAWKDCENC*CREG*EKVSRQAYYRVETLFGT**
 * SPRCSHY*RKS*RTSSSCGHLRIRYSICKEIPHRQRPNTNGGGRRAKDSCLRYRNPLSRR **
 * RRVWKRPNYND*LCR*K*SKGDYLEKHRSSIR*GCIKRERDDKEISQDYQGEGS*HYSYL **
 * *WRLIRLPTFSEKGRKTWD*INHWKRWKRAQDAENRRYDGCRSXGKNTFRLVSCNNKDNK **
 * SPXIHTRGCI*XNFWKA **


 * <span style="font-family: Arial,sans-serif; font-size: 10pt;">Translate results **
 * >rf 2 Untitled **
 * FCLTLRRRYTMGHHHHHHHHHHSSGHIEGRHMILDVDYITEEGKPVIRLFKKENGKFKIE **
 * HDRTFRPYIYALLRDDSKIEEVKKITGERHGKIVRIVDVEKVEKKFLGKPITVWKLYLEH **
 * PQDVPTIREKVREHPAVVDIFEYDIPFAKRYLIDKGLIPMEGEEELKILAFDIETLYHEG **
 * EEFGKGPIIMISYADENEAKVITWKNIDLPYVEVVSSEREMIKRFLRIIREKDPDIIVTY **
 * NGDSFDFPHLAKRAEKLGIKLTIGRDGSEPKMQRIGDMTAVEVXGRIHFDLYHVITRTIN **
 * LPXYTLEAVYEXIFGKP **

SQXTH*RLYMXQFLESX
 * <span style="font-family: Arial,sans-serif; font-size: 10pt;">Translate results **
 * >rf 3 Untitled **
 * FV*L*EGDIPWAIIIIIIIIITAAAISKVVI*F*MWIT*LKKENLLLGYSKKRTENLR*S **
 * MIELLDHTFTLFSGMIQRLKKLRK*RGKGMERL*ELLM*RRLRKSFSASLLPCGNFIWNI **
 * PKMFPLLEKKLENIQQLWTSSNTIFHLQRDTSSTKA*YQWRGKKS*RFLPSI*KPSITKE **
 * KSLEKAQL **LVMQMKMKQR*LLGKT*IFHTLRLYQARER**RDFSGLSGRRILTL*LLI**
 * METHSTSHI*RKGQKNLGLN*PLEEMEASPRCRE*AI*RL*KSXEEYISTCIM **QGQ*I

<span style="color: #222222; font-family: Arial,sans-serif; font-size: 11.5pt; line-height: 1.5;">DNA polymerase [Pyrococcus furiosus] <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">Sequence ID: ref|WP_011011325.1| Length: 775Number of Matches: 1 <span style="color: #336699; font-family: Arial,sans-serif; font-size: 10pt;">[|See 9 more title(s)]

Query 32 MILDVDYITEEGKPVIRLFKKENGKFKIEHDRTFRPYIYALLRDDSKIEEVKKITGERHG 91 MILDVDYITEEGKPVIRLFKKENGKFKIEHDRTFRPYIYALLRDDSKIEEVKKITGERHG Sbjct 1 MILDVDYITEEGKPVIRLFKKENGKFKIEHDRTFRPYIYALLRDDSKIEEVKKITGERHG 60
 * <span style="font-family: Verdana,sans-serif; font-size: 1pt;">Alignment statistics for match #1 ||
 * **<span style="color: #606060; font-family: Verdana,sans-serif; font-size: 7.5pt;">Score ** || **<span style="color: #606060; font-family: Verdana,sans-serif; font-size: 7.5pt;">Expect ** || **<span style="color: #606060; font-family: Verdana,sans-serif; font-size: 7.5pt;">Method ** || **<span style="color: #606060; font-family: Verdana,sans-serif; font-size: 7.5pt;">Identities ** || **<span style="color: #606060; font-family: Verdana,sans-serif; font-size: 7.5pt;">Positives ** || **<span style="color: #606060; font-family: Verdana,sans-serif; font-size: 7.5pt;">Gaps ** ||
 * <span style="font-family: Verdana,sans-serif; font-size: 9pt;">566 bits(1459) || <span style="font-family: Verdana,sans-serif; font-size: 9pt;">0.0 || <span style="font-family: Verdana,sans-serif; font-size: 9pt;">Compositional matrix adjust. || <span style="font-family: Verdana,sans-serif; font-size: 9pt;">282/286(99%) || <span style="font-family: Verdana,sans-serif; font-size: 9pt;">283/286(98%) || <span style="font-family: Verdana,sans-serif; font-size: 9pt;">0/286(0%) ||
 * **<span style="color: #606060; font-family: Verdana,sans-serif; font-size: 7.5pt;">Score ** || **<span style="color: #606060; font-family: Verdana,sans-serif; font-size: 7.5pt;">Expect ** || **<span style="color: #606060; font-family: Verdana,sans-serif; font-size: 7.5pt;">Method ** || **<span style="color: #606060; font-family: Verdana,sans-serif; font-size: 7.5pt;">Identities ** || **<span style="color: #606060; font-family: Verdana,sans-serif; font-size: 7.5pt;">Positives ** || **<span style="color: #606060; font-family: Verdana,sans-serif; font-size: 7.5pt;">Gaps ** ||
 * <span style="font-family: Verdana,sans-serif; font-size: 9pt;">566 bits(1459) || <span style="font-family: Verdana,sans-serif; font-size: 9pt;">0.0 || <span style="font-family: Verdana,sans-serif; font-size: 9pt;">Compositional matrix adjust. || <span style="font-family: Verdana,sans-serif; font-size: 9pt;">282/286(99%) || <span style="font-family: Verdana,sans-serif; font-size: 9pt;">283/286(98%) || <span style="font-family: Verdana,sans-serif; font-size: 9pt;">0/286(0%) ||

Query 92 KIVRIVDVEKVEKKFLGKPITVWKLYLEHPQDVPTIREKVREHPAVVDIFEYDIPFAKRY 151 KIVRIVDVEKVEKKFLGKPITVWKLYLEHPQDVPTIREKVREHPAVVDIFEYDIPFAKRY Sbjct 61 KIVRIVDVEKVEKKFLGKPITVWKLYLEHPQDVPTIREKVREHPAVVDIFEYDIPFAKRY 120

Query 152 LIDKGLIPMEGEEELKILAFDIETLYHEGEEFGKGPIIMISYADENEAKVITWKNIDLPY 211 LIDKGLIPMEGEEELKILAFDIETLYHEGEEFGKGPIIMISYADENEAKVITWKNIDLPY Sbjct 121 LIDKGLIPMEGEEELKILAFDIETLYHEGEEFGKGPIIMISYADENEAKVITWKNIDLPY 180

Query 212 VEVVSSEREMIKRFLRIIREKDPDIIVTYNGDSFDFPHLAKRAEKLGIKLTIGRDGSEPK 271 VEVVSSEREMIKRFLRIIREKDPDIIVTYNGDSFDFP+LAKRAEKLGIKLTIGRDGSEPK Sbjct 181 VEVVSSEREMIKRFLRIIREKDPDIIVTYNGDSFDFPYLAKRAEKLGIKLTIGRDGSEPK 240

Query 272 MQRIGDMTAVEVXGRIHFDLYHVITRTINLPXYTLEAVYEXIFGKP 317 MQRIGDMTAVEV GRIHFDLYHVITRTINLP YTLEAVYE IFGKP Sbjct 241 MQRIGDMTAVEVKGRIHFDLYHVITRTINLPTYTLEAVYEAIFGKP 286

Day 29 (7/29/14): We measured the absorbency level of the culture until it was 0.3 using Chipper. This only took an hour which is fishy. Then we added IPTG and had fun. We FINISHED endnote. Woop woop party time. We also got our page gel back from Friday. These are the final results:

Day 30 (7/30/14): We finished making the PFU by centrifuging, purifying, and storing the samples. Then we nanodropped the samples.

Day 31 (7/31/14): This is our last day :( *Tear* We are making a PCR of the PFU to make sure it works.

=Week 8=

Day 23 (7/21/14): We ran a PCR with the samples of PFU stored at different temperatures. We did this to find which storage was best for maintaining the PFU. (I apologize for the bad picture quality and orientation) Figure 1. 1% Agarose Lane 1: 1kb ladder Lane 2: 0,06 pNIC with PFU 4C Lane 3: 0.6 pNIC with PFY 4C Lane 4: 6 pNIC with PFU 4C Lane 5: 0.06 pNIC with PFU -20C/glycerol Lane 6: 0.6 pNIC with PFU -20C/glycerol Lane 7: 6 pNIC with PFU -20/glycerol Lane 8: 1kb ladder This PCR shows no result from the PCR. Before running ANOTHER PCR, we decided to test whether or not the PFU had degraded.

Day 24 (7/22/14): We ran some Nanodrops of the PFU. The original concentration was 1.09mg/ml. These results show a dramatic rise in concentration. We concluded that the PFU had broken into pieces to cause that rise, but in reality that doesn't make sense because it is the same amount of PFU. It could have been that some of the buffer evaporated, leaving a higher concentration of PFU in comparison to amount of buffer solution.

Day 25 (7/23/14): We began to make a page gel to see whether or not the PFU actually degraded. We forgot to put in the dye, so we couldnt' insert the solutions into the wells.

Day 26 (7/24/14): We made ANOTHER page gel. We had to try a couple times. The first time, it didn't solidify. The second, one of them leaked. At the end of the day, we had one gel (a perfect gel, might i add). We wrapped it in damp kimwipes and stored it in the 4C fridge overnight.

Day 27 (7/25/14): We ran the samples on the page gel using the gel we made the previous day. This isn't the final result, but you can tell that there are multiple bands, indicating that the PFU had in fact degraded. We have to make it again! Fun! :) I guess we know what we are doing next week.

=Week 7= Great work Sarah! - Dr. B 072114

Day 19 (7/15/14): (Missed Friday and Monday *sad face*) We transformed the calcium competent cells we had made the previous week. Sample A: 1ng DNA, 15 ul bacteria. Three colonies grown. Sample B: 5ng DNA, 15ul bacteria. 130 colonies grown..about. Sampe C: 25ng DNA, 15ul bacteria. 300 colonies grown...about. This indicates that our Ca competent cells are competent.

Day 20 (7/16/14): We did a PCR of using pNIC and Taq and also one of pNIC and PFU Polyemerase in order to see if we made it correctly. We then ran a gel. Figure 1: 1% Agarose Lane 1: 100bp ladder. Lane 2: 0.06 pNIC using Taq Lane 3: 0.6 pNIC using Taq Lane 4: 6 pNIC using Taq Lane 5: 0.06 pNIC using PFU Lane 6: 0.6 pNIC using PFU Lane 7: 6 pNIC using PFU This gel indicates an unsuccessful PCR with both samples. We started the PCR 5 degrees below the starting point as directed and also had the PFU work under the same conditions as Taq. So we will have to run another PCR of the pNIC using Taq and will immediately begin at the starting temperature. We will also run another PCR using the PFU Polymerase with the proper conditions.

Day 21 (7/17/14): We found the conditions necessary to run a PCR using PFU Polymerase. In order to do this, we referenced several sources including several published articles. In one article [Insert Link here] the authors ran a PCR with the following conditions: 95C for 30s, 95C for 5s, 55C for 1min, 72C for 2.5min, 4C forever. We compared this to the conditions for Q5 which are as follows: 95C for 3min, 98C for 10s, 55C for 30s, 72C for 2.5min, 4C forever. Because the major difference is the second temperature [find out if elongation or denatuation], we ran a PCR with a temperature gradient from 92C to 100C. Figure 2 (actually, it's like figure 29...but who's counting?): 1% Agarose Lane1: 100 bp ladder Lane 2: 10 pNIC PFU at 100C Lane 3: 10 pNIC PFU at 99C Lane 4: 10 pNIC PFU at 98C Lane 5: 10 pNIC PFU at 97C Lane 6: 10 pNIC PFU at 96C Lane 7: 10 pNIC PFU at 95C Lane 8: 10 pNIC PFU at 94C Lane 9: 10 pNIC PFU at 93C This PCR indicates that the proper conditions for PFU would be either 100C or 98C. So, we have to do another one with each of those conditions.

Day 22 (7/18/19): We did two PCRs with two different conditions for PFU (100C and 98C). Figure 3: 1% Agarose Lane 1: 100 bp ladder Lane 2: 0.06 pNIC PFU at 98C Lane 3: 0.6 pNIC PFU at 98C Lane 4: 6 pNIC PFI at 98C Lane 5: 0.06 pNIC PFU at 100C Lane 6: 0.6 pNIC PFU at 100C Lane 7: 6 pNIC PFU at 100C This PCR indicates that the 98C is the proper condition. Woop woop. We stored the PFU at -20C with 50% glycerol.

=Week 6= Good work. You're slowly becoming actual scientists :D -Grace (07/15/14) ___ Day 14 (7/7/14): We did nickel purification of the PFU polymerase. We then nanodropped the two eluted samples. This concentration is 1.09mg/ml which is a good concentration indicating a successful sample. We nanodropped this sample and found the concentration to be 0.05. We knew that this sample would have a lower concentration because it was eluted twice, but because this concentration was so drastically different, we nanodropped again and got 0.07. We wanted some consistency, so we decided to nanodrop once more and got a concentration of 0.06 which was between our first two, so we decided that was about the right concentration. We also began the protocol for competent cells by streaking out frozen glycerol stock of bacterical cells onto an LB plate without antibiotics.

Day 15 (7/8/14): We made overnight cultures of PFU Polymerase in BL21 DE3 so we could acquire a plasmid sample of the PFU Polymerase. We also made an overnight culture using the plates grown up the night before. Then, we made solutions of CaCl2, MgCl2, CaCl2 15% glycerol and autoclaved it. Photography by Braxton Schultz

Day 16 (7/9/14): We Midi Prepped the plasmid from the overnight culture and nanodropped it to find the concentration which was 103.9ng/ul. Photography by Roshni Ray We then transferred the overnight culture of competent cells to two flasks of 500 ml of LB. We then began measuring the OD600 every hour until it reached 0.35. After completing this step in the protocol, we realized that we had another 4 hour procedure to do. Rats. So we decided to repeat this procedure the next day in order to do the entire 8 hour procedure. woop woop.

Day 17 (7/10/14): We did the last 8 hour part of the competent cell protocol. Also, we sent the plasmids of the PFU Polymerase off to sequencing.

=Week 5=

Day 10 (6/30/14): We did restriction enzyme digest on the plasmids we Midi Prepped on the Thursday of the previous week (6/26/14). These are the solutions prepared for running the gel. We used an uncut plasmid as "control" to compare the other cut bands to.

Day 11 (7/1/14): We ran a gel of our "digested" plasmids. Restriction Enzyme Digest result. 1% Agarose, IKB. Lane 1-Ladder. Lane 2-uncut plasmid Braxton. Lane 3-uncut plasmid Sarah. Lane 4-EcoRI Braxton. Lane 5-PvuII Braxton. Lane 6-EcoRI and PvuII Braxton. Lane 7-EcoRI Sarah. Lane 8-PvuII Sarah. Lane 9-EcoRI and PvuII Sarah. This gel was successful (?). I put a question mark because there are streaks in the gel, but also the picture is bad quality. However the following is true: the EcoRI has the same number of bases/same band location as the uncut plasmid which makes sense because the "circle" was only cut once (on Braxton's it is higher because it was supercoiled when uncut). The PvuII has two bands because that enzyme cut the plasmid twice, creating two pieces, one larger and one smaller. The EcoRI and PvuII has three bands because the plasmid was cut three times, creating three pieces. The brightness of the band is dimmer if the band is smaller because it does not contain as much Ethidium bromide which helps illuminate it in the UV light.

Day 12-13 (7/2/14-7/3/14): We began making PFU Polymerase. We grew a 4 ml overnight culture in LB + AMP (This step was actually done on 7/1/14). The next day, we incubated the cells and measured the absorbance. We needed to start measuring the OD600 every hour when the absorbance was 0.05 (that would be the starting absorbance), however we couldn't get it there. So, we added all of our overnight culture to one flask and let it incubate until after the documentary when we would add IPTG. On Thursday, we lysed the cells (breaked open the cell to allow the protein come out) and completed the initial purification of the cells (this separated the protein from the other junk by spinning it down in the centrifuge). Then we stored it overnight for the long weekend! Happy Birthday America :)

=**Week 4**=

Day 5 (6/23/14): We analyzed a DNA sequence. First, we did a BLAST of our sequence in order to compare the sequence to certain genomes to determine what kind of organism this DNA came from. We found that this was in fact a type of coral, however there was a mutation (one base pair was off). Next, we translated the original sequence into a protein and decided which frame to use by determining which frame had the longest sequence of amino acids (the longest protein). Then we compared protein translations of the Purple Protein and the protein from the original sequence. We did a BLAST to confirm that the mutation previously mentioned did not affect the resulting protein. It indeed did not. We then inserted the gene into a vector backbone (pGEMT) and located some important segments of nucleotides such as the Start codon, the Shine-Dalgamo sequence, the Stop codon, and the M13 Reverse primer site. After completing this sequence of pGBR22, we cut it using the restriction enzymes EcoRI and PvuII. Then we made an overnight culture using a colony from the plates made on Friday.

Day 6 (6/24/14): We did a Midi Prep using the culture prepared that Monday. Super fun. Then I nanodropped those my sample and found its concentration to be 176.3ng/ul. Then we sent all of our samples to the DNA Sequencing Core and called it a day.

Day 7 (6/25/14): We nanodropped the plasmid samples that we had Midi Prepped the day before. My concentration was around 170ng/ul which indicates a successful sample. We then sent that sample to the DNA Sequencing Core. [Insert picture here] We then made 5 liters of LB including one liter with agarose. We used that liter to make 25 plates: 10 with Amp and 15 with Kan. (Or how Martin pronounces it, "Kahn").

Day 8 (6/26/14): We were supposed to do restriction enzyme digest today, but the plasmids were left out overnight. Instead, we took our extra flasks of cultures and Midi Prepped them before sending them off to be sequenced. This concentration is about double that of my previous plasmid because we used 500ul of the TE Buffer during Midi Prep instead of 1000ul.

Day 9 (6/27/14): We ran a gel of the degraded plasmids to see the extent of the damage done.

=**Week 3**=

Day 1 (6/17/14): We did the Pipettor Calibration Lab in order to learn how to calibrate micropipettors on an analytical balance and to evaluate precision and accuracy of these measurements. We used P1000, P200, and P20 pipettors to measure microscopic amounts which is a mandatory skill needed in the lab. The calculated values of the standard deviation, percent errors, and coefficient of variations were graphed in order to illustrate the accuracy and precision of each pipettor. From this data, it can be concluded that the P20 was the most accurate, followed closely by the P1000. However, the P1000 is clearly the most precise.

Day 2 (6/18/14): We did the Buffers and Solutions lab using the measuring skills obtained the previous day. We made seven solutions because the materials for the eighth were not found in the lab. This lab required multiple calculations that will be commonly used in future labs. This also allowed us an opportunity to learn where everything is in the lab and how to use certain materials such as a pH probe and vacuum filter.

Day 3 (6/19/14): We finished the Buffers and Solutions lab before learning how to Nanodrop. We nanodropped a plasmid with a known concentration in order to know if we did the protocol successfully. This plasmid had a concentration of 170ng/ul. Therefore my measured concentration of 169.4ng/ul shows that I correctly nanodropped. Yay. We then learned how to send a plasmid off to DNA Sequencing. using option B (we inserted the primer into the solution). We used a 10uM primer which we had to obtain by diluting a 100uM primer. After adding the primer, plasmid, and water, filled out a DNA Sequencing Request form and took the samples to the ICMB core facility.

Day 4 (6/20/14): We transformed bacteria with plasmid DNA. We used DH5alpha (E. Coli) as our bacteria and pGBR22 as the plasmid.