Jiaqi+Z.

Week 11&12 The next immediate step is to harvest by centrifugation and lysis probably on the next day.
 * More results would be good. virtual work? -UM**

Protocol Protein Expression Scaled Up:

Large Culture Step: 10ml of the sample from previous tubes were transferred into 500ml of LB media, which was prepared about few days during the same week. Then, Vernier Visible Spec was used and OD600 was measured every 30 minutes until it reached around 0.5. Then sample 0 and sample 1 were taken and were grow on shaker for 18-20 hours at ~25 degree Celsius.

In Day 2, option A was chosen. 6 colonies were picked for 6 separate tubes instead of 5 because this leaves some space if some errors occurred. After 8 hrs proceeded to large culture step.

In Day1, the plasmid with our positive clone was transformed with BL21 (DE3) cells. Two separate plates were used. Below were the transformed bacteria on the next day. 10 ul of plasmid was transformed with 50ul BL21 cells on the plate. Some colonies were appeared after 1 day.

50ul of plasmid was transformed with 50ul BL21 cells on the plate. Barely any colonies appeared. Even this plate did not grow much colonies, the 10ul sample was ok and could be used for next step.

More LB media with plates were preped just in case if the supplies ran out.

After the third trial of cloning, I still could not get a good concentration, which was very similar to the first and second result. I was trying to figure out my pitfalls during cloning process. One of my partners got a positive clone and was already sequenced, so I proceeded onto the next step, which is the transformation.

Week 9&10
 * Overall good job. Missing captions for images. - BN**

However, one of our group member actually got a positive clone. If my third trial run bad again, the group's positive clones could be used for later steps.

Preparation of pNIC-Bsa4 as Accepting Vector In my first trial and second trial, my results' concentration were not good. I even did a gel check to see if this could cut and I got a blank image. I have been stuck on this process for some days. This image could be resulted from some contamination, such as dH2O was used versus Nanopure. A third trial should be conducted in the next week.


 * Protocol LB Medium **

goal was to make more medium for the bacteria to grow just in case if the medium runs out.


 * Protocol LB Agar Kan + Suc plates **

Those plates were also preped for my group members as backup.

Purpose is to extract DNA from transformed bacterial cells The protocol from the kit was used as guide in this lab. In my first trial, one of the TA told me the wrong information, which turned out that I eluted in the wrong column. I later confirmed with another TA, which worked out this time on my second trial. However, this whole process took very long time. On my second trial, the result was good: The concentration was 28.2 ng/ul, which was fine.
 * Protocol Midi Prep Kit - HiSpeed **

Week 7&8 Nice concentration on PCR Squared! More data/lab work would be good. -UM The Next step is to mini-prep the pellets and be ready for cloning. Then one colony was picked and putted into media with Kan antibiotics. The next day the pellets were collected. The pellets obtained after centrifuge for 15 minutes. This is one of the tubes. I performed 4 tubes.

After the PCR^2 products were confirmed to be successful and the concentration was good, we proceeded to preparing LB media and culturing bacteria and to transform them. This is the transformed bacteria with Kan antibiotics. Cultured in the plate.

Elution of my PCR squared products were performed and results were obtained. First trial was not successful The first trail did not give a high concentration. There was probably a error performed in the elution process and spectrophotometry step. I redid this again.

This is the second trial of elution with spectrophotometry which is pretty successful and this give a higher concentration. This also proved that my PCR^2 was very successful.

PCR^2 reaction actually took a longer time than expected because when I came back to obtain the overnight solutions from the machine, those were gone so I have to redo the whole process again. This took a longer time to get to the washing step.

Crop images and include image of first trial of PCR. Good job! - Michael T. Week 5 & 6: I am very sorry that I misunderstand about the wikispaces. At the first time, I was uploading all the stuffs onto googledocs and after a few weeks realized that this place is where I upload stuffs. I am really sorry.


 * PCR squared reaction was also performed with the designed tails on Saturday and left overnight. After clean up, I can start preparing cloning on Monday.**


 * Primary & Secondary PCR:**

This is using our group's designed tails for the Secondary PCR, which was successful and this showed that our tails designed successfully. Lane1 was the 100bp DNA ladder. Lane2 was a smear, which is the primary PCR, as predicted because there was no primers. Lane3 was our secondary PCR, the actual product obtained.

First time the primary and secondary PCR were not successful and I redid these, which took a longer time to finish my second trial. I think the first time there was a human error.

Forward Primer Tail: 5’ _TAC TTC CAA TCC ATG AAG AAT TGG GTT AAA GTT _ 3’ _ bp GC Content 33.3_%
 * PCR Primer Design Tails for pNIC-Bsa4 cloning **

0 mM Mg2+ Tm 57.1_ oC 1.5 mM Mg2+ Tm _65.1 oC 2 mM Mg2+ Tm _67.4 oC 4 mM Mg2+ Tm 67_ oC 6 mM Mg2+ Tm 67.5_ oC

Reverse Primer Tail: Reverse Primer: 5’ _ TATCCACCTTTACTGTTAGTACAGGTACGC 3’ _ bp

GC Content _43.3%

0 mM Mg2+ Tm 60.4_ oC 1.5 mM Mg2+ Tm 68_ oC 2 mM Mg2+ Tm _67.1 oC

4 mM Mg2+ Tm _69.6 oC 6 mM Mg2+ Tm _70.1 __oC__

Week 3 & 4 Results ? - 092713


 * Primer Dilutions for Assembly Step **

Goal: to make an Oligo Mix consisting of all of our primers that were ordered after designing them in DNA works. The stock concentrations are 100uM and the final concentration should be 1uM for each primer. Our gene of interest has 22 Oligos, so we added 78 ul nanopure H2O for the dilution. This will be for the PCR.


 * Restriction Enzyme Digest:**

__ Results: __ In lane2 is the 1kb DNA ladder, which is like a standard for the template. Lane 3 is the uncat plasmid, in this image, there is one band that is showning. Lane 4 is the sample1 with EcoRi digestions, sample 1 appears to be heavier than the uncut plasmid because the sample1 has a higher band than uncut plasmid, which indicates that it moves slower than uncut plasmid. Lane 5 is sample2 with PvuII digestions, it has two bands and both of them are lighter than digestion with only EcoRI. Lane 6 is both of EcoRI and PvuII combined, this image is very similar to the two individual combined. It has three bands, which indicates cutting three times and formed three bands.
 * Goal ** : digest pGBR22 plasmid with restriction enzymes and visualize fragments on gel. Basically, we are digesting with three digestion reactions. First one is EcoRI, second one is PyuII, and third one is EcoRI + PyuII, which is a double digestion.

Generally, the bands appeared as expected because EcoRI cuts one time, PuVII cuts two times, so when they combined together, there should be three bands, as indicated by this gel image. However, there could be something went wrong. For example, if not storing some of the samples in the ice bucket, some of the DNA can break their bonds and affact the result.


 * PCR pmCherry results:**

Lane1 looks like a smear and this is the DNA ladder. I skipped one lane and proceed to the next lane for #1 PCR solution. Threrefore lane 3,4,5, 6 were PCR solutions with VDSR1&2 primers. Lane 7,8,9 were PCR solutions with M13 Forward & Reverse Primers. Both results were very similar and there is a small gap in lane 9, which could be due to human error. Lane 3 was very obscure and this could also due to human error. My mistakes from week1&2 might be due to the amount of volume that I added were so insignificant, which could be the reason that I always get blank image. This trial was the third trial of this same gene.

__Week 1 & 2__ __Results ? - 090913__

Goal: was to amplify the pmcherry protein. My results were not successful in the first and second week. All the results I got was blank. I redo this in week 3&4.
 * PCR pmCherry**


 * DNA Sequencing **

the goal was to to determine and analyze the DNA sequence of a specific plasmid. Human genomic plus transcript (Human G+T) results:   

 Analysis/Conclusion: We can also use the sequencing machine several times to do the larger DNA sequence. For example, for 1500bp, we can do 750bp from the left direction and 750bp from the right direction. Then, look for the overlap region and eliminate it. Throughout this experiment, the researcher learned the way to determine and analyze the DNA sequence in a specific plasmid, such as comparing and contrast the Query Covers and cutting with various digesting enzymes. The next step is to design specific primers that were useful in many PCR labs or perform similar sequencing to analyze more DNA.


 * Oligo Primer Design:**

goal: design an oligo set of forward and reverse primers for PCR synthesizing and amplifying the Protein Tyrosine Phosphatase - lmo1800 gene so that it can be inserted into a cloning (or expression) vector. 22 oligonucleotides need to be synthesized   1 ATGAAAAATTGGGTTAAAGTTACCGGTGCTGGTGTTCTGAGCGCAACCCTGCTGC 55  2 CATTCGCTTCCGCTTTCTCTTCAGACTGCGCACCACAACCACCCAGCAGCAGGGTTGCGC 60  3 AGAGAAAGCGGAAGCGAATGTTAAAACCGAGCAAACCCTGAAACCTGGTTCTCAGATCAA 60  4 AACCGCCCAGGTCACGAACATTCACCGCACCTTCCAGCTTGATCTGAGAACCAGGTTTCA 60  5 CGTGACCTGGGCGGTTACAAAACCACCGACGGTCTGACTATTAAACCGCACAAACTGATC 60  6 GTCGCTGTCAGACAGGTTCGCCAGTTCCGCGCTACGGATCAGTTTGTGCGGTTTAATAGT 60  7 GAACCTGTCTGACAGCGACAAGAAGAAACTGGTTAACACCTACGACCTGTCCCACATCGT 60  8 GGGTCAGGTTTAGTCGCAACTTCAGAAGAGGTACGAAAATCGACGATGTGGGACAGGTCG 60 <span style="background-color: #ffffff; font-family: 'Courier New',Courier,monospace; font-size: 14px;"> 9 TTGCGACTAAACCTGACCCAAAACTGACCGACGTTGACTACACCCACGATTCTGTTATGA 60 <span style="background-color: #ffffff; font-family: 'Courier New',Courier,monospace; font-size: 14px;">10 CGGTGAGGTCCTGGGTAGAGGTGCTAGTACCGTTGTCTTTCATAACAGAATCGTGGGTGT 60 <span style="background-color: #ffffff; font-family: 'Courier New',Courier,monospace; font-size: 14px;">11 CTACCCAGGACCTCACCGCGTCTCTCGCGAAGATGGACAACCCGGAAACCTTTCTGATCA 60 <span style="background-color: #ffffff; font-family: 'Courier New',Courier,monospace; font-size: 14px;">12 TGAATGCTCGTCTCATCCGTGATGAAGCTCTTATTAGCGTTGATCAGAAAGGTTTCCGGG 60 <span style="background-color: #ffffff; font-family: 'Courier New',Courier,monospace; font-size: 14px;">13 CGGATGAGACGAGCATTCAGGCGTACAAAGACTTCTTCGACATCCTGCTCGCAAATCAGG 60 <span style="background-color: #ffffff; font-family: 'Courier New',Courier,monospace; font-size: 14px;">14 ACGATCCTTACCCGCGGTGCAGTGCCAGAGAACGGAACCGTCCTGATTTGCGAGCAGGAT 60 <span style="background-color: #ffffff; font-family: 'Courier New',Courier,monospace; font-size: 14px;">15 CCGCGGGTAAGGATCGTGCCGGTTTCGGTACGGCGCTCGTTCTGTCTGCCCTCGGCGTTG 60 <span style="background-color: #ffffff; font-family: 'Courier New',Courier,monospace; font-size: 14px;">16 GTATTTGTTAGACAGCATGTAATCGTCAATAACGGTGTTTTTGTCAACGCCGAGGGCAGA 60 <span style="background-color: #ffffff; font-family: 'Courier New',Courier,monospace; font-size: 14px;">17 CGATTACATGCTGTCTAACAAATACCGTGCGGACGAAAACAAAAAGGCGATCGAAGCGGT 60 <span style="background-color: #ffffff; font-family: 'Courier New',Courier,monospace; font-size: 14px;">18 CGTCATACCATCGATTACCTTCTTATTGTCGGTCTTCGCCGCTACCGCTTCGATCGCCTT 60 <span style="background-color: #ffffff; font-family: 'Courier New',Courier,monospace; font-size: 14px;">19 AGAAGGTAATCGATGGTATGACGGCGGTAATGGAAGTGCGTGAATCTTACATTAACGCGG 60 <span style="background-color: #ffffff; font-family: 'Courier New',Courier,monospace; font-size: 14px;">20 ATCCATAGAACCGTATTTCGCATTAATCTCGTCGAACGCCGCGTTAATGTAAGATTCACG 60 <span style="background-color: #ffffff; font-family: 'Courier New',Courier,monospace; font-size: 14px;">21 TGCGAAATACGGTTCTATGGATAACTTCCTCAAAGAAAAACTGGGTCTCACTGATGCCAA 60 <span style="background-color: #ffffff; font-family: 'Courier New',Courier,monospace; font-size: 14px;">22 TTAGTACAGGTACGCTTTTTTCAGCTGTTCTTTTTTGGCATCAGTGAGACCCA 53

<span style="font-family: Arial,sans-serif; font-size: 10pt;">my group member ordered this for <span style="background-color: #ffffff; font-family: Arial,Helvetica,sans-serif; line-height: 1.5;">Listeria monocytogenes