Ruifei+W.


 * 12/06/2012**

Final results of enzyme assay for PSTP were decent but irregular (sorry had to screenshot- Linux is weird):

Graph of PSTP inhibition as a function of inhibitor 5154121 concentration. The normalization refers to the subtracting each value from the no enzyme control with 0.11 mM inhibitor (thus, 0.11 mM actually has two bars – one that is shown and one that is 0). The trend is discontinuous, but there appears to be a downwards shift of activity with increasing enzyme concentration. The orange bar indicates another negative control trial with just enzyme. The enzyme PSTP actually appears to be fully functional in this trial as opposed to the enzyme assay.



Graph of PSTP absorbance for enzyme assay. The positive control shows up well, which is indicative of a non-functional enzyme.


 * 11/27/2012**

Enzyme assay on YopH (purple tube B) today. Results:

LoggerPro data graph for first trial of enzyme assay of YopH. The highest absorbance value was for tube F at a value of 0.150 (which is still very low compared to the 2.259 value obtained for the positive control last time).

Analysis:

Not sure if YopH dimerizes, but it is almost 8 months old. In addition, the diluted version was used (1 ng/uL) which may have reduced its efficacy. Another source of error may have been that I forgot to pipette up and down during the initial 10 minute incubation period.

112612 - Good - but include some 'data' or images. Dr B other enzyme is 'YopH' Performed enzyme assay again today unsuccessfully (did PNP positive control which turned out very well though). Will move onto using the YopH protein next week for further enzyme assay.
 * 11/20/2012**

LoggerPro data graph for second trial of enzyme assay of PSTP with included positive control (PNP). The PNP curve is orange and had an absorbance of 2.259 at 410 nm which is far above any other absorbance value recorded.

Analysis:

The PSTP is probably polymerized and thus non-functional. Will move onto YopH.

Ruifei - probably need to use someone else's enzyme. Either PSTP or FtHap. -- DR. B 11/19/12
 * 11/15/2012**

Did enzyme assay unsuccessfully - all values were in the noisy range. Possible errors: dimerization, bad PNPP, user error. Will go faster next time.


 * 11/08/2012-11/09/2012**

Suman did FPLC. Results:





Analysis:

Direct nanodrop concentrations are a bit low.


 * 11/07/2012**

Planning on doing FPLC tomorrow.

Finishing up protein characterization (gel looks good so far! just have to dry it):

10/29/2012 Ruifei W, Brandon N, Suman A PSTP protein gel Lane 1; Skip Lane 2; NEB Protein Ladder Lane 3; Sample 5 trial 1 Lane 4; Sample 6 trial 1 Lane 5; Sample 7 (elution 1) trial 1 Lane 6; Sample 8 (elution 1) trial 1 Lane 7; Sample 5 trial 2 Lane 8; Sample 6 trial 2 Lane 9; Sample 7 (elution 1) trial 2 Lane 10; Sample 8 (elution 1) trial 2

Analysis:

 Elution 1 trial 1 yields two bands, around 25 and 65 kDaltons. Elution 1 trial 2 yields only one band at around 25 kDaltons. Based on these results, the samples were combined for FPLC. Elution 2 for both trials yielded no protein which means that elution theoretically captured all the protein. Samples 5 and 6 look normal.

Performing concentration of protein to 1 mL (combined samples 1 and 2 of Elution since the gel bands looked good). Concentration using nanodrop:



Concentrations are around 3.735 mg/mL in a 1 mL concentration making the yield (pre-FPLC): **3.735 mg.**


 * 11/06/2012**

Sonication, purification performed today on both tubes as two trials. TCEP was added pre-sonication as well as to the two elutions.

Tomorrow or Thursday, we will perform protein gel characterization as well as FCLP.


 * 11/05/2012**

Tomorrow, will perform sonication/purification with both tubes of pellet and an addition 48 uL of TCEP to each 12 mL lysis buffer tube pre-sonication to prevent protein dimerization.

Successfully made 5th/5th from last tail primers secondary PCR (image not shown); however, will move onto cloning Sadhana's target PSTP.


 * 10/29/2012**

Tried option A secondary PCR with both first/last primers from oligo as well as third/third from last primers from oligo (in addition to normal tail end primers) at 58 Celsius. Also present in this gel are the concatenated results of pNIC cutting. Gel results: 10/29/2012 Ruifei W <span style="font-family: 'Times New Roman',serif;">Ruifei's Secondary PCR Results for Oligonucleotide mix for serine threonine phosphatase (STP) and pNIC RE Digest <span style="font-family: 'Times New Roman',serif;">Lane 1; Skip <span style="font-family: 'Times New Roman',serif;">Lane 2; 1 kb NEB DNA Ladder <span style="font-family: 'Times New Roman',serif;">Lane 3; 100 bp NEB DNA Ladder <span style="font-family: 'Times New Roman',serif;">Lane 4; 5 uL of pNIC RE digest combined sample <span style="font-family: 'Times New Roman',serif;">Lane 5; 5 uL of tail end primer secondary PCR at 58 Celsius <span style="font-family: 'Times New Roman',serif;">Lane 6; 5 uL of first/last oligo secondary PCR at 58 Celsius <span style="font-family: 'Times New Roman',serif;">Lane 7; 5 uL of third/third from last oligo secondary PCR at 58 Celsius <span style="font-family: 'Times New Roman',serif;">Lanes 8-10; Tom's samples


 * 10/25/2012**

Checked validity of DNAWorks output...we seem to have gotten the wrong protein sequence. What we used for protein sequence: http://www.ncbi.nlm.nih.gov/protein/AAL58473.1

What I think we should have used: http://www.ncbi.nlm.nih.gov/protein/2PK0_D

The difference is only by 5 amino acids, but the 2PK0 actually exists on the PDB whereas the protein we are cloning now is not on the PDB. Also, it was from 2003 while the 2PK0 is from 2012 (more recent).

Ran primary and secondary PCR with new reagents (hopefully will obtain better results). Primary at 57 Celsius and secondary at 58 Celsius. Also ran 4 pNIC cutting trials to see if they cut correctly.

<span style="font-family: 'Times New Roman',serif;">10/25/2012 <span style="font-family: 'Times New Roman',serif;">Ruifei W <span style="font-family: 'Times New Roman',serif;">Ruifei's Primary and Secondary PCR Results for Oligonucleotide mix for serine threonine phosphatase (STP) and pNIC RE Digest <span style="font-family: 'Times New Roman',serif;">Lane 1; Skip <span style="font-family: 'Times New Roman',serif;">Lane 2; 1 kb NEB DNA Ladder <span style="font-family: 'Times New Roman',serif;">Lane 3; 100 bp NEB DNA Ladder <span style="font-family: 'Times New Roman',serif;">Lane 4; 5 uL of primary PCR at 57 Celsius <span style="font-family: 'Times New Roman',serif;">Lane 5; 5 uL of secondary PCR at 58 Celsius <span style="font-family: 'Times New Roman',serif;">Lane 6; 5 uL of pNIC RE digest at 6.6 ng/uL <span style="font-family: 'Times New Roman',serif;">Lane 7; 5 uL of pNIC RE digest at 7.7 ng/uL <span style="font-family: 'Times New Roman',serif;">Lane 8; 5 uL of pNIC RE digest at 7.8 ng/uL <span style="font-family: 'Times New Roman',serif;">Lane 9; 5 uL of pNIC RE digest at 10.75 ng/uL


 * 10/23/2012**

Ran primary PCR at 57 Celsius, secondary at 58 Celsius. Gel results:

<span style="font-family: 'Times New Roman',serif;">10/24/2012 <span style="font-family: 'Times New Roman',serif;">Ruifei W <span style="font-family: 'Times New Roman',serif;">Ruifei's and Primary Secondary PCR Results for Oligonucleotide mix for serine threonine phosphatase (STP) <span style="font-family: 'Times New Roman',serif;">Lane 1; Skip <span style="font-family: 'Times New Roman',serif;">Lane 2; 100 bp NEB DNA Ladder <span style="font-family: 'Times New Roman',serif;">Lane 3; 5 uL of primary PCR at 57 Celsius <span style="font-family: 'Times New Roman',serif;">Lane 4; 5 uL of secondary PCR at 58 Celsius

<span style="font-family: 'Times New Roman',serif;">**Analysis:**

<span style="font-family: 'Times New Roman',serif;">It appears that the primary PCR has a thick band around 450 bp as does the secondary PCR. The size of my gene is closer to 750 bp, so the PCR did not appear to work.

102112 -Ruifei, ok not sure what is going on with this. Try the newer kit to see if that helps and be sure you have right elution solution. - Dr. B Nanodrop results for pNIC cutting (made sure that elution solution was added to center of elution column):
 * 10/19/2012**



Analysis:

The nanodrop results continue to be disappointing - the exact reason for the low concentration of cut pNIC is not clear but could be attributed to not pipetting the elution solution to the center of the column.

Secondary results PCR for 49, 51, and 53 Celsius:

<span style="font-family: 'Times New Roman',serif;">10/19/2012 <span style="font-family: 'Times New Roman',serif;">Ruifei W <span style="font-family: 'Times New Roman',serif;">Ruifei's Secondary PCR Results for Oligonucleotide mix for serine threonine phosphatase (STP) <span style="font-family: 'Times New Roman',serif;">Lane 1; Skip <span style="font-family: 'Times New Roman',serif;">Lane 2; 100 bp NEB DNA Ladder <span style="font-family: 'Times New Roman',serif;">Lane 3; 5 uL of secondary PCR at 49 Celsius <span style="font-family: 'Times New Roman',serif;">Lane 4; 5 uL of secondary PCR at 51 Celsius <span style="font-family: 'Times New Roman',serif;">Lane 5; 5 uL of secondary PCR at 53 Celsius


 * 10/17/2012**

pNIC concentration again too low - results from that below:

Analysis:

It appears that there may be something going on to take the pNIC out of solution (perhaps during the column PCR clean-up step). I will try centrifuging with 12000g instead of 16000g next time.

Also, gel shows that my primary PCR worked. My secondary from last time, turned out to have a 150 bp band which is definitely not the size of my gene.

<span style="font-family: 'Times New Roman',serif;">10/17/2012 <span style="font-family: 'Times New Roman',serif;">Ruifei W <span style="font-family: 'Times New Roman',serif;">Ruifei's Cut pNIC and Secondary PCR Results for Oligonucleotide mix for serine threonine phosphatase (STP)/Rishi's Results <span style="font-family: 'Times New Roman',serif;">Lane 1; Skip <span style="font-family: 'Times New Roman',serif;">Lane 2; 100 bp NEB DNA Ladder <span style="font-family: 'Times New Roman',serif;">Lane 3; 10 uL of primary PCR <span style="font-family: 'Times New Roman',serif;">Lane 4: 5 uL of secondary PCR at 58 Celsius(from last time) <span style="font-family: 'Times New Roman',serif;">Lanes 5-rest: Rishi's

<span style="font-family: 'Times New Roman',serif;">Analysis: The primary PCR seems to have worked as a smear appeared. With this result, I will proceed to secondary PCR tomorrow. In addition, I will attempt the pNIC cut again tomorrow.


 * 10/16/2012**

pNIC cutting failed again because I didn't realize I had to put the pNIC in the center of column. I will try again tomorrow along with the PCR.





Got the gel results:

<span style="font-family: 'Times New Roman',serif;">10/16/2012 <span style="font-family: 'Times New Roman',serif;">Ruifei W <span style="font-family: 'Times New Roman',serif;">Ruifei's Cut pNIC and Secondary PCR Results for Oligonucleotide mix for serine threonine phosphatase (STP)/Aldo's Results <span style="font-family: 'Times New Roman',serif;">Lane 1; Skip <span style="font-family: 'Times New Roman',serif;">Lane 2; 1 kb NEB DNA Ladder <span style="font-family: 'Times New Roman',serif;">Lane 3; 5 uL of cut pNIC-Bsa4 with EcoRI <span style="font-family: 'Times New Roman',serif;">Lane 4: 12 uL of secondary PCR at 58 Celsius <span style="font-family: 'Times New Roman',serif;">Lane 5: 12 uL of secondary PCR at 59 Celsius <span style="font-family: 'Times New Roman',serif;">Lane 6: 12 uL of secondary PCR at 60 Celsius <span style="font-family: 'Times New Roman',serif;">Lanes 7-10: Aldo's results

<span style="font-family: 'Times New Roman',serif;">**Analysis:**

<span style="font-family: 'Times New Roman',serif;"> It definitely appears that the secondary PCR at the default 58 Celsius worked because the band appears between the 0.5 and 1 kb base pair mark (my gene is 750 bp). In addition, the pNIC cut successfully but the concentration was too low. Thus, my next step is to continue with PCR^2 and then try cutting the pNIC to a higher concentration. ^Never mind - I only saw what I wanted to see. The band for secondary is probably just primer dimers. I will try again from primary PCR.

First, I will first check the concentration of my pNIC in the freezer before using for cutting.

pNIC purple tube:

260/280: 1.99, 260/230: 2.65, conc: **172.8 ng/uL**

pNIC orange tube:

260/280: 1.99, 260/230: 2.76, conc: **161.8 ng/uL**


 * Analysis:**

There is nothing wrong with the concentrations of my plasmid - there must have been something wrong with my preparation of the cutting of pNIC. I will try again later today.

101612 - Ruifei - these pNIC values seem too low to work with. You may want to make more. Potentially we could have soemone make some - but if you have the time - go ahead. -- Dr. B
 * 10/11/2012:**

Will gel check the secondary PCR as well as the cut pNIC tomorrow.

Prepared pNIC-Bsa4 accepting vector with the following nanodrop results:





Average concentration of pNIC-Bsa4 accepting vector: **10.7 ng/uL**. Relatively low concentration. DNA stored in -20 fridge.

Trying primary PCR again. Gel results: <span style="font-family: 'Times New Roman',serif;">10/11/2012 <span style="font-family: 'Times New Roman',serif;">Ruifei W <span style="font-family: 'Times New Roman',serif;">Primary PCR Results for Oligonucleotide mix for serine threonine phosphatase (STP) <span style="font-family: 'Times New Roman',serif;">Lane 1; Skip <span style="font-family: 'Times New Roman',serif;">Lane 2; 1 kb NEB DNA Ladder <span style="font-family: 'Times New Roman',serif;">Lane 3; 12 uL of oligonucleotide mix primary PCR

Analysis:

The primary PCR seems to have a larger thick band at around 450 bp instead of a smear - it should be ok for secondary PCR though. Will try secondary PCR later today.


 * 10/10/2012:**

Secondary PCR failed again. Primary PCR left for too long in the PCR machine - possible mistake from that. Will try it again tomorrow. Gel results:

<span style="font-family: 'Times New Roman',serif;">10/10/2012 <span style="font-family: 'Times New Roman',serif;">Ruifei W <span style="font-family: 'Times New Roman',serif;">Primary and Secondary PCR Results for Oligonucleotide mix for serine threonine phosphatase (STP) <span style="font-family: 'Times New Roman',serif;">Lane 1; Skip <span style="font-family: 'Times New Roman',serif;">Lane 2; 1 kb NEB DNA Ladder <span style="font-family: 'Times New Roman',serif;">Lane 3; 12 uL of oligonucleotide mix primary PCR <span style="font-family: 'Times New Roman',serif;">Lane 4; 12 uL of oligonucleotide mix secondary PCR annealed at 50 Celsius <span style="font-family: 'Times New Roman',serif;">Lane 5; 12 uL of oligonucleotide mix secondary PCR annealed at 52 Celsius <span style="font-family: 'Times New Roman',serif;">Lane 6; 12 uL of oligonucleotide mix secondary PCR annealed at 54 Celsius <span style="font-family: 'Times New Roman',serif;">Lane 7; 12 uL of oligonucleotide mix secondary PCR annealed at 58 Celsius


 * Analysis:**

The oligo mix was made again and it seems that the primary PCR worked mostly well with a dark spot that could indicate contamination. The secondary PCR did not show up which could be the result of:

1) annealing temperature 2) bad primer dilution (I doubt that though)


 * 10/09/2012:**

Failure may have been due to

1) incorrectly made oligo mix (will have to make again anyways - out of primary PCR) 2) incorrect annealing temperature (forward primer addition from CDS Tm ~ 48, reverse primer addition from CDS Tm ~ 49.5)

Why not do annealing temp of entire primer?

Perhaps another variable to try is annealing temperature.

Started first virtual screening refresher run.

Performed primer dilution and initiated secondary PCR. Gel results:

<span style="font-family: 'Times New Roman',serif;">10/09/2012 <span style="font-family: 'Times New Roman',serif;">Ruifei W <span style="font-family: 'Times New Roman',serif;">Primary and Secondary PCR Results for Oligonucleotide mix for serine threonine phosphatase (STP) <span style="font-family: 'Times New Roman',serif;">Lane 1; Skip <span style="font-family: 'Times New Roman',serif;">Lane 2; 1 kb NEB DNA Ladder (bled over a little to lane 1) <span style="font-family: 'Times New Roman',serif;">Lane 3; 15 uL of oligonucleotide mix primary PCR <span style="font-family: 'Times New Roman',serif;">Lane 4; 15 uL of oligonucleotide mix secondary PCR

Analysis: The secondary PCR did not work. Will re-make oligo-mix and start primary/secondary PCR process over.

100912 - ok looks good Ruifei.
 * 10/04/2012:**

Second time trying primary PCR: (successful - see lane 2)

<span style="font-family: 'Times New Roman',serif;">10/04/2012 <span style="font-family: 'Times New Roman',serif;">Ruifei W <span style="font-family: 'Times New Roman',serif;">Two Trials of Primary PCR Results for Oligonucleotide mix for serine threonine phosphatase (STP) <span style="font-family: 'Times New Roman',serif;">Lane 1; Skip <span style="font-family: 'Times New Roman',serif;">Lane 2; 1 kb NEB DNA Ladder (bled over a little to lane 1) <span style="font-family: 'Times New Roman',serif;">Lane 3; 15 uL of trial 1 oligonucleotide mix primary PCR <span style="font-family: 'Times New Roman',serif;">Lane 4; 15 uL of trial 2 oligonucleotide mix primary PCR

Analysis:

This time, it definitely seems as though the primary PCR worked thoroughly (the product is now in the -20 fridge). The gel once again illustrates that the first primary PCR attempt has a very weak if existent band in lane 1. Because a new batch of KOD polymerase was used for trial 2, that may have fixed the issue with the primary PCR. Next, I will move onto primer dilution, secondary PCR, and pNIC-Bsa4 excising.

Earlier in the day:

Tried primary and secondary run in a single gel - it seemed to fail though. <span style="font-family: 'Times New Roman',serif;">10/04/2012 <span style="font-family: 'Times New Roman',serif;">Ruifei W <span style="font-family: 'Times New Roman',serif;">Primary and Option A (using first and last primers in oligo mix instead of tail primers) Secondary PCR Results for Oligonucleotide mix for serine threonine phosphatase (STP) <span style="font-family: 'Times New Roman',serif;">Lane 1; Skip <span style="font-family: 'Times New Roman',serif;">Lane 2; 1 kb NEB DNA Ladder <span style="font-family: 'Times New Roman',serif;">Lane 3; 15 uL of oligonucleotide mix primary PCR <span style="font-family: 'Times New Roman',serif;">Lane 4; 15 uL of oligonucleotide mix option A secondary PCR

<span style="font-family: 'Times New Roman',serif;">Analysis:

<span style="font-family: 'Times New Roman',serif;">There does not seem to be any assembled gene in the gel. Possible sources of error: error in PCR or gel was run too long and DNA escaped (unlikely because ladder is still present). <span style="font-family: 'Times New Roman',serif;">It is strange that the previous gel run (see below on 09/28/2012) had only 5 uL of primary PCR result show up, but this gel couldn't even show 15 uL of primary PCR result. Possible degradation of primary/secondary PCR results? Potentially because they may have been left out of the -20 freezer for a while the gel was being set up.

<span style="font-family: 'Times New Roman',serif;">**10/03/2012:**

Did option A secondary PCR with first and last oligos in the wells (B10 and D3). Tried to run gel, but accidentally used 50x TAE when preparing the gel and so the samples did not run properly. Will try again tomorrow.


 * 09/28/2012:**

Finished PyMol refresher.

Did a practice primary PCR with oligo mix:

<span style="font-family: 'Times New Roman','serif';">9/28/2012 <span style="font-family: 'Times New Roman','serif';">Ruifei W <span style="font-family: 'Times New Roman','serif';">Primary PCR Results for Oligonucleotide mix for serine threonine phosphatase (STP) <span style="font-family: 'Times New Roman','serif';">Lane 1; Skip <span style="font-family: 'Times New Roman','serif';">Lane 2; 1 kb NEB DNA Ladder <span style="font-family: 'Times New Roman','serif';">Lane 3; 1 kb NEB DNA Ladder <span style="font-family: 'Times New Roman',serif;">Lane 4; 5 uL of oligonucleotide mix post primary PCR

<span style="font-family: 'Times New Roman','serif';">The result of primary PCR is one smeared band, which is what the lab protocol predicted. The band is smaller because only 5 uL of primary PCR result was added.

100112 - Rufei, ok there are 2 start codons in your primer sequence. Your 2nd PCR looks good. You are good to move forward on your cloning. Did you already to Oligo Mix? - Dr. B
 * 09/27/2012:**

Made 100 uL of 1 uM oligo mix from 18 STP wells in primer box. <span style="font-family: 'Times New Roman','serif';">0.305 ng of DNA <span style="background-color: transparent; color: #000000; font-family: Arial; font-size: 15px; text-decoration: none; vertical-align: baseline;">Forward primer for STP: TACTTCCAATCCATG <span style="background-color: transparent; color: #000000; font-family: Arial; font-size: 15px; vertical-align: baseline;">__ATGGAAATCTCTCTCCTC__ <span style="background-color: transparent; color: #000000; font-family: Arial; font-size: 15px; text-decoration: none; vertical-align: baseline;">Reverse primer for STP: TATCCACCTTTACTG <span style="background-color: #ff0000; color: #000000; font-family: Arial; font-size: 15px; vertical-align: baseline;">__TTA__ <span style="background-color: transparent; color: #000000; font-family: Arial; font-size: 15px; vertical-align: baseline;">__TTCTTCGTCCGTCAC__

Forward Primer corrected for extra ATG: <span style="background-color: transparent; color: #000000; font-family: Arial; font-size: 15px; text-decoration: none; vertical-align: baseline;">TACTTCCAATCC <span style="background-color: transparent; color: #000000; font-family: Arial; font-size: 15px; vertical-align: baseline;">__ATGGAAATCTCTCTCCTC__

<span style="background-color: transparent; color: #000000; font-family: Arial; font-size: 11px; text-decoration: none; vertical-align: baseline;">Table 1: GC content and melting temperature as a function of the concentration of Mg++ for forward and reverse primers for insertion of S. agalactiae serine/threonine phosphatase gene into pNIC-Bsa4 vector with 0.4 uM Oligo concentration, 50 mM Na+ concentration, 0.3 mM dNTP concentration
 * || <span style="background-color: transparent; color: #000000; font-family: Arial; font-size: 15px; text-decoration: none; vertical-align: baseline;">GC Content || <span style="background-color: transparent; color: #000000; font-family: Arial; font-size: 15px; text-decoration: none; vertical-align: baseline;">T(m) at 0 mM Mg++ (°C) || <span style="background-color: transparent; color: #000000; font-family: Arial; font-size: 15px; text-decoration: none; vertical-align: baseline;">T(m) at 1.5 mM Mg++ (°C) || <span style="background-color: transparent; color: #000000; font-family: Arial; font-size: 15px; text-decoration: none; vertical-align: baseline;">T(m) at 2 mM Mg++ (°C) || <span style="background-color: transparent; color: #000000; font-family: Arial; font-size: 15px; text-decoration: none; vertical-align: baseline;">T(m) at 4 mM Mg++ (°C) || <span style="background-color: transparent; color: #000000; font-family: Arial; font-size: 15px; text-decoration: none; vertical-align: baseline;">T(m) at 6 mM Mg++ (°C) ||
 * <span style="background-color: transparent; color: #000000; font-family: Arial; font-size: 15px; text-decoration: none; vertical-align: baseline;">Forward primer || <span style="background-color: transparent; color: #000000; font-family: Arial; font-size: 15px; text-decoration: none; vertical-align: baseline;">42.4% || <span style="background-color: transparent; color: #000000; font-family: Arial; font-size: 15px; text-decoration: none; vertical-align: baseline;">60.7 || <span style="background-color: transparent; color: #000000; font-family: Arial; font-size: 15px; text-decoration: none; vertical-align: baseline;">68.1 || <span style="background-color: transparent; color: #000000; font-family: Arial; font-size: 15px; text-decoration: none; vertical-align: baseline;">68.6 || <span style="background-color: transparent; color: #000000; font-family: Arial; font-size: 15px; text-decoration: none; vertical-align: baseline;">69.6 || <span style="background-color: transparent; color: #000000; font-family: Arial; font-size: 15px; text-decoration: none; vertical-align: baseline;">70.0 ||
 * <span style="background-color: transparent; color: #000000; font-family: Arial; font-size: 15px; text-decoration: none; vertical-align: baseline;">Reverse Primer || <span style="background-color: transparent; color: #000000; font-family: Arial; font-size: 15px; text-decoration: none; vertical-align: baseline;">42.4% || <span style="background-color: transparent; color: #000000; font-family: Arial; font-size: 15px; text-decoration: none; vertical-align: baseline;">61.0 || <span style="background-color: transparent; color: #000000; font-family: Arial; font-size: 15px; text-decoration: none; vertical-align: baseline;">68.3 || <span style="background-color: transparent; color: #000000; font-family: Arial; font-size: 15px; text-decoration: none; vertical-align: baseline;">68.8 || <span style="background-color: transparent; color: #000000; font-family: Arial; font-size: 15px; text-decoration: none; vertical-align: baseline;">69.8 || <span style="background-color: transparent; color: #000000; font-family: Arial; font-size: 15px; text-decoration: none; vertical-align: baseline;">70.3 ||

<span style="background-color: transparent; color: #000000; font-family: Arial; font-size: 11px; text-decoration: none; vertical-align: baseline;">Table 2: GC content and melting temperature as a function of the concentration of Mg++ for **CORRECTED** forward and reverse primers for insertion of S. agalactiae serine/threonine phosphatase gene into pNIC-Bsa4 vector with 0.4 uM Oligo concentration, 50 mM Na+ concentration, 0.3 mM dNTP concentration


 * || <span style="background-color: transparent; color: #000000; font-family: Arial; font-size: 15px; text-decoration: none; vertical-align: baseline;">GC Content || <span style="background-color: transparent; color: #000000; font-family: Arial; font-size: 15px; text-decoration: none; vertical-align: baseline;">T(m) at 0 mM Mg++ (°C) || <span style="background-color: transparent; color: #000000; font-family: Arial; font-size: 15px; text-decoration: none; vertical-align: baseline;">T(m) at 1.5 mM Mg++ (°C) || <span style="background-color: transparent; color: #000000; font-family: Arial; font-size: 15px; text-decoration: none; vertical-align: baseline;">T(m) at 2 mM Mg++ (°C) || <span style="background-color: transparent; color: #000000; font-family: Arial; font-size: 15px; text-decoration: none; vertical-align: baseline;">T(m) at 4 mM Mg++ (°C) || <span style="background-color: transparent; color: #000000; font-family: Arial; font-size: 15px; text-decoration: none; vertical-align: baseline;">T(m) at 6 mM Mg++ (°C) ||
 * <span style="background-color: transparent; color: #000000; font-family: Arial; font-size: 15px; text-decoration: none; vertical-align: baseline;">Forward primer || 43.3 || 59.5 || 66.7 || 67.2 || 68.3 || 68.8 ||
 * <span style="background-color: transparent; color: #000000; font-family: Arial; font-size: 15px; text-decoration: none; vertical-align: baseline;">Reverse Primer || <span style="background-color: transparent; color: #000000; font-family: Arial; font-size: 15px; text-decoration: none; vertical-align: baseline;">42.4% || <span style="background-color: transparent; color: #000000; font-family: Arial; font-size: 15px; text-decoration: none; vertical-align: baseline;">61.0 || <span style="background-color: transparent; color: #000000; font-family: Arial; font-size: 15px; text-decoration: none; vertical-align: baseline;">68.3 || <span style="background-color: transparent; color: #000000; font-family: Arial; font-size: 15px; text-decoration: none; vertical-align: baseline;">68.8 || <span style="background-color: transparent; color: #000000; font-family: Arial; font-size: 15px; text-decoration: none; vertical-align: baseline;">69.8 || <span style="background-color: transparent; color: #000000; font-family: Arial; font-size: 15px; text-decoration: none; vertical-align: baseline;">70.3 ||

<span style="background-color: transparent; color: #000000; display: block; font-family: Arial; font-size: 15px; text-align: justify; text-decoration: none; vertical-align: baseline;">Combined FULL pNIC-Bsa4 plasmid with STP insertion (start codon ATG highlighted in neon green, forward primer highlighted in olive green, stop codon TAA highlighted in red, tail primer (complemented from above) highlighted in turquoise, and 6xHis tags highlighted in pink):

**<span style="color: #000000; font-family: 'Arial','sans-serif'; font-size: 11.3333px;">Combined FULL pNIC-Bsa4 plasmid with STP insertion (start codon ATG highlighted in neon green, forward primer highlighted in olive green, main STP sequence highlighted in yellow, stop codon TAA highlighted in red, tail primer (complemented from above) highlighted in turquoise, and 6xHis tags highlighted in pink): **

<span style="font-family: 'Arial','sans-serif'; font-size: 11.3333px;">TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATG CACCATCATCATCATCAT <span style="color: #000000; font-family: 'Arial','sans-serif'; font-size: 11.3333px;">TCTTCTGGTGTAGATCTGGGTACCGAG <span style="color: #000000; font-family: 'Arial','sans-serif'; font-size: 11.3333px;">AACCTG TACTTCCAATCCATG ATG GAAATCTCTCTCCTCACCGACATCGGCCAGCGTCGTAGCAACAATCAGGACTTCATTAACCAATTCGAAAACAAAGCGGGTGTTCCGCTGATCATCCTGGCGGATGGTATG GGCGGTCACCGTGCGGGTAATATCGCGTCTGAGATGACCGTTACCGACCTGGGTTCTGATTGGGCGGAGACCGATTTTTCCGAGCTGTCTGAAATCCGTGACTGGATGCTGGTTAGCATCGAAACGGAGAACCG TAAAATCTATGAACTCGGTCAGAGCGACGACTATAAAGGCATGGGTACCACCATCGAAGCAGTTGCCATTGTTGGCGATAACATCATTTTCGCGCACGTTGGTGACTCTCGTATCGGTATCGTACGTCAAGGCG AATACCACCTGCTGACTTCTGACCACTCTCTGGTAAACGAACTCGTTAAAGCAGGCCAGCTGACTGAAGAGGAAGCGGCGTCTCACCCGCAGAAGAACATTATCACCCAATCTATTGGCCAGGCTAACCCAGT TGAACCGGACCTCGGTGTGCACCTCCTGGAGGAAGGTGACTACCTGGTCGTCAACTCCGACGGCCTCACCAATATGCTCTCTAACGCGGACATCGCGACCGTGCTGACCCAAGAAAAAACCCTGGATGACAAA AACCAAGACCTGATCACCCTGGCGAACCATCGTGGTGGTCTGGACAACATCACCGTTGCTCTGGTTTACGTTGAATCCGAGGCAGTCTCCCTCGAACTGCTGGACGAACGTATGACGGTTCGTTCTGTTAAAGAA TGCTGGAAAGGTGGTTCTACGGCAGTGTGCTGCGCGATCGACATGGACCAGAAACTGATGGCGCTCGCCTGGCTCGGTGATTCTCCGGGTTACGTTATGTCTAACATCGAATTCCGTCAGCTCACTCGTGGTCAC TCTCCGAGCGATGAGCGCGAAGCGCGTCGTGTAGAAGAAGCCGGTGGCCAACTCTTCGTTATTGGCGGTGAACTGCGTGTTAACGGTGTTCTGAACCTGACCCGCGCACTCGGCGACGTTCCGGGTCGTCCGA TGATCTCTAATGAGCCGGAAACCTGCCAGGTTCCAATCGAGTCTTCCGATTATCTCGTCCTGCTGGCTTGCGACGGTATTTCTGACGTATTCAATGAGCGTGACCTGTATCAGCTCGTAGAGGCCTTTGCCAACG ACTACCCAGTGGAAGATTACGCGGAACTGTCTCGCTTCATCTGCACCAAAGCGATTGAGGCGGGCTCTGCGGACAACGTTTCTGTTGTGATCGGTTTCCTGCGTCCACCGCAGGATGTGTGGAAACTCATGAA ACATGAATCTGATGATGAGGATTCCGACGTGACGGACGAAGAA TAA CAGTAAAGGTGGATA <span style="color: #000000; font-family: 'Arial','sans-serif'; font-size: 11.3333px;">CGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAG CACCACCACCACCACCAC <span style="color: #000000; font-family: 'Arial','sans-serif'; font-size: 11.3333px;">TGAGA <span style="color: #000000; display: block; font-family: Arial,sans-serif; font-size: 11.3333px; text-align: justify;">TCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGA <span style="color: #000000; display: block; font-family: Arial,sans-serif; font-size: 11.3333px; text-align: justify;">TTGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTT <span style="color: #000000; display: block; font-family: Arial,sans-serif; font-size: 11.3333px; text-align: justify;">CGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGG <span style="color: #000000; display: block; font-family: Arial,sans-serif; font-size: 11.3333px; text-align: justify;">TTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAA <span style="color: #000000; display: block; font-family: Arial,sans-serif; font-size: 11.3333px; text-align: justify;">ATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCG <span style="color: #000000; display: block; font-family: Arial,sans-serif; font-size: 11.3333px; text-align: justify;">CTCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCC <span style="color: #000000; display: block; font-family: Arial,sans-serif; font-size: 11.3333px; text-align: justify;">ATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAG <span style="color: #000000; display: block; font-family: Arial,sans-serif; font-size: 11.3333px; text-align: justify;">AATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCG <span style="color: #000000; display: block; font-family: Arial,sans-serif; font-size: 11.3333px; text-align: justify;">CTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGT <span style="color: #000000; display: block; font-family: Arial,sans-serif; font-size: 11.3333px; text-align: justify;">GGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAG <span style="color: #000000; display: block; font-family: Arial,sans-serif; font-size: 11.3333px; text-align: justify;">AAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGA <span style="color: #000000; display: block; font-family: Arial,sans-serif; font-size: 11.3333px; text-align: justify;">CGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAA <span style="color: #000000; display: block; font-family: Arial,sans-serif; font-size: 11.3333px; text-align: justify;">AGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGA <span style="color: #000000; display: block; font-family: Arial,sans-serif; font-size: 11.3333px; text-align: justify;">GCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGT <span style="color: #000000; display: block; font-family: Arial,sans-serif; font-size: 11.3333px; text-align: justify;">CTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCT <span style="color: #000000; display: block; font-family: Arial,sans-serif; font-size: 11.3333px; text-align: justify;">ATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGT <span style="color: #000000; display: block; font-family: Arial,sans-serif; font-size: 11.3333px; text-align: justify;">TTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCG <span style="color: #000000; display: block; font-family: Arial,sans-serif; font-size: 11.3333px; text-align: justify;">TTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCC <span style="color: #000000; display: block; font-family: Arial,sans-serif; font-size: 11.3333px; text-align: justify;">TTACGCATCTGTGCGGTATTTCACACCGCATATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGC <span style="color: #000000; display: block; font-family: Arial,sans-serif; font-size: 11.3333px; text-align: justify;">CAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGG <span style="color: #000000; display: block; font-family: Arial,sans-serif; font-size: 11.3333px; text-align: justify;">TAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCT <span style="color: #000000; display: block; font-family: Arial,sans-serif; font-size: 11.3333px; text-align: justify;">GTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGG <span style="color: #000000; display: block; font-family: Arial,sans-serif; font-size: 11.3333px; text-align: justify;">TAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACAT <span style="color: #000000; display: block; font-family: Arial,sans-serif; font-size: 11.3333px; text-align: justify;">AATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATT <span style="color: #000000; display: block; font-family: Arial,sans-serif; font-size: 11.3333px; text-align: justify;">CATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGATCATGCGCACCCGTGGGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGG <span style="color: #000000; display: block; font-family: Arial,sans-serif; font-size: 11.3333px; text-align: justify;">ACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTAC <span style="color: #000000; display: block; font-family: Arial,sans-serif; font-size: 11.3333px; text-align: justify;">GAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCT <span style="color: #000000; display: block; font-family: Arial,sans-serif; font-size: 11.3333px; text-align: justify;">AACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCA <span style="color: #000000; display: block; font-family: Arial,sans-serif; font-size: 11.3333px; text-align: justify;">CCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGA <span style="color: #000000; display: block; font-family: Arial,sans-serif; font-size: 11.3333px; text-align: justify;">GCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCT <span style="color: #000000; display: block; font-family: Arial,sans-serif; font-size: 11.3333px; text-align: justify;">CATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAA <span style="color: #000000; display: block; font-family: Arial,sans-serif; font-size: 11.3333px; text-align: justify;">CTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATA <span style="color: #000000; display: block; font-family: Arial,sans-serif; font-size: 11.3333px; text-align: justify;">ACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTT <span style="color: #000000; display: block; font-family: Arial,sans-serif; font-size: 11.3333px; text-align: justify;">CGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCG <span style="color: #000000; display: block; font-family: Arial,sans-serif; font-size: 11.3333px; text-align: justify;">CCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCAT <span style="color: #000000; display: block; font-family: Arial,sans-serif; font-size: 11.3333px; text-align: justify;">ACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATG <span style="color: #000000; display: block; font-family: Arial,sans-serif; font-size: 11.3333px; text-align: justify;">CGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCC <span style="color: #000000; display: block; font-family: Arial,sans-serif; font-size: 11.3333px; text-align: justify;">GAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGA <span style="color: #000000; display: block; font-family: Arial,sans-serif; font-size: 11.3333px; text-align: justify;">GATCTCGATCCCGCGAAAT


 * 09/24/2012:**

Primer design for pNIC-Bsa4 cloning

Rufie - not super bright bands - but good enough. This PCR doesn't seem to work as well as the first one does. -- DR. B Did the pLIC sequencing vector of pNIC-Bsa4 PCR. Gel result:
 * 09/20/2012**:

<span style="font-family: 'Times New Roman','serif';">9/20/2012 <span style="font-family: 'Times New Roman','serif';">Ruifei W <span style="font-family: 'Times New Roman','serif';">PCR Results for pNIC-Bsa4 sequenced with pLIC-for and pLIC-rev <span style="font-family: 'Times New Roman','serif';">Lane 1; Skip <span style="font-family: 'Times New Roman','serif';">Lane 2; 100 bp NEB DNA Ladder <span style="font-family: 'Times New Roman','serif';">Lane 3; 0.0305 ng of DNA <span style="font-family: 'Times New Roman','serif';">Lane 4; 0.305 ng of DNA <span style="font-family: 'Times New Roman','serif';">Lane 5; 3.05 ng of DNA <span style="font-family: 'Times New Roman','serif';">Lane 6; Control - no DNA


 * Analysis:**The PCR results turned out as expected. From Lanes 3-5, the bands got noticeable wider in size (Lane 3 unfortunately, is barely visible so it is hard to discern whether there is simply a small amount of amplified gene, or none). There was no visible band in Lane 6 as expected, which means that there was no contamination this time in the PCR (as compared to last time when there was). Finally, the size of the gene amplified matches up because the SacB gene is longer than 1517 bp, which is the maximum that the 100 bp ladder can measure. It seems reasonable the distance the amplified genes traveled up the gel. <span style="font-family: 'Calibri','sans-serif'; font-size: 14.6667px;">Overall, it seems that the CDS region was amplified correctly using the pLIC primers.

Got results from DNA sequencing. Did a side-by-side BLAST comparison with the sequencing results from the first DNA sequencing of pNIC-Bsa4 and also a BLAST comparison for the forward/reverse against each other:

1. Comparison of pLIC-for against first DNA sequencing: 79% query coverage, Max Identity = 99% 2. Comparison of pLIC-rev against first DNA sequencing: 79% query coverage, Max Identity = 99% 3. Comparison of pLIC-for against pLIC-rev: no significant query coverage

Result of (3.) is predictable because the same thing happened with the first pLIC-for/pLIC-rev comparison. This is due to the fact that the gene that the primers surround (SacB) is longer than 1000 bp so the primers going forward and reverse will not meet in the middle.

The query coverage being 79% is fairly good, indicating that the Midi-prep yielded mostly pNIC-Bsa4.


 * 09/18/2012:**

Submitted DNA from Midi-prep to DNA sequencing.

Ruifei - good job - include brief analysis or your gel result. -- Dr. B091812
 * 09/14/2012:**

Midi-prep of Bsa4 product from transformation.

Trial 2 screenshot from Nanodrop:




 * whoops - accidentally spilled half of my final DNA sample while nanodropping ( -.- ") *

Average concentration: 163.5 ng/uL Average 260/280: 2.09 Average 260/230: 2.76


 * 09/13/2012:**

PCR Gel Result:



<span style="font-family: 'Times New Roman','serif';">9/13/2012 <span style="font-family: 'Times New Roman','serif';">Ruifei W <span style="font-family: 'Times New Roman','serif';">Primary PCR Results for pGBR22 DNA <span style="font-family: 'Times New Roman','serif';">Lane 1; Skip <span style="font-family: 'Times New Roman','serif';">Lane 2; 100 bp NEB DNA Ladder <span style="font-family: 'Times New Roman','serif';">Lane 3; 0.3 ng of DNA <span style="font-family: 'Times New Roman','serif';">Lane 4; 3 ng of DNA <span style="font-family: 'Times New Roman','serif';">Lane 5; 30 ng of DNA <span style="font-family: 'Times New Roman','serif';">Lane 6; Control - no DNA

<span style="font-family: 'Times New Roman','serif';">**Analysis:** There was a slight error in the gel running because the control lane (6) has some DNA in it, indicating possible contamination. In addition, the band thicknesses do not increase from 3 -5 regularly - it increases from lane 3-4 and then seems to decrease slightly from lanes 4-5. This may be due to errors while running the gel.


 * 09/11/2012:**

Spun down pNIC-Bsa4 transformed plates (which grew successfully) and stored products in -20 degrees freezer.


 * 09/07/2012:**

Performed analysis of DNA sequence of pGEMT plasmid; results of virtual RE digest below:

<span style="font-family: Times New Roman,serif;">Virtual Restriction Digest of pGBR22

<span style="font-family: Times New Roman,serif;">Lane 1; 1 kb NEB DNA Ladder

<span style="font-family: Times New Roman,serif;">Lane 2; EcoRI

<span style="font-family: Times New Roman,serif;">Lane 3; PvuII

<span style="font-family: Times New Roman,serif;">Lane 4; EcoRI/PvuII <span style="font-family: Times New Roman,serif;">__**Question:**__ <span style="font-family: Times New Roman,serif;">How would you determine what your target sequence was if it is bigger than the 1000 <span style="font-family: Times New Roman,serif;">bp limit of the DNA Sequencing machines? (That is how we got the sequence in the <span style="font-family: Times New Roman,serif;">beginning of this exercise) <span style="font-family: Times New Roman,serif;">Answer: The target sequence is first sliced into multiple pieces and each piece is individually sequenced.

Also, did Day 2 of transformation (started LB culture). Plates were successful.


 * 09/07/2012:**

Transformed bacteria and stored plates at 4 degrees.


 * 09/06/2012:**



<span style="font-family: 'Times New Roman','serif';">9/6/2012 <span style="font-family: 'Times New Roman','serif';">Ruifei W <span style="font-family: 'Times New Roman','serif';">Restriction Digest of pGBR22 <span style="font-family: 'Times New Roman','serif';">Lane 1; Skip <span style="font-family: 'Times New Roman','serif';">Lane 2; 1 kb NEB DNA Ladder <span style="font-family: 'Times New Roman','serif';">Lane 3; Uncut pGBR22 plasmid <span style="font-family: 'Times New Roman','serif';">Lane 4; EcoRI-Hf <span style="font-family: 'Times New Roman','serif';">Lane 5; PvuII <span style="font-family: 'Times New Roman','serif';">Lane 6; EcoRI-Hf/PvuII

<span style="font-family: 'Times New Roman','serif';">**Analysis:** The gels run here turned out almost exactly like the virtual gel digest predicted that it would. That is, one band for EcoRI, two bands for PvuII, and three bands for PvuII + EcoRI.


 * Target (protein/gene name):** fructose-1,6-bisphosphate aldolase
 * NCBI Gene # or RefSeq#:** <span class="wiki_link_ext">PF3D7_1444800
 * Protein ID (NP or XP #) or Wolbachia#:**P14223 (from [])
 * Organism:** //Plasmodium falciparum//
 * Etiologic Risk Group (see link below):** RG2
 * Background/Disease Information:** P. falciparum is a protozoan parasite that is instrumental in causing malaria - the number one killer infectious disease in the world. The fructose-1,6-bisphosphate aldolase enzyme is a glycolytic enzyme which if targeted, could halt the production of ATP for the protozoan which would effectively kill it. The p. falciparum version of the enzyme also differs from the human version of the enzyme making selective targeting possible.
 * Essentiality of this protein:** Affects glycolysis so it is very essential to p. falciparum survivial (link to article: [])
 * Complex of enzymes:** no
 * EC#:** 4.1.2.13
 * Link to BRENDA EC# page:** []
 * --** Show screenshot of BRENDA enzyme mechanism schematic


 * [[image:http://www.chem.qmul.ac.uk/iubmb/enzyme/reaction/polysacc/polygif/41213.gif caption="4.1.2.13 Reaction Mechanism http://www.chem.qmul.ac.uk/iubmb/enzyme/reaction/polysacc/41213.html"]] ||
 * 4.1.2.13 Reaction Mechanism http://www.chem.qmul.ac.uk/iubmb/enzyme/reaction/polysacc/41213.html ||


 * Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):**
 * -- link to Sigma (or other company) page for assay or assay reagents (substrates)**
 * -- link to paper that contains assay information:** []
 * -- List cost and quantity of substrate reagents and supplier**

From __Sigma__: (1) Trizma base, 25 g for $15.40 (2) D-Fructose 1,6-Diphosphate, Tetra(cyclohexylammonium) Salt, 5 g for $102.00 (3) ß-Nicotinamide Adenine Dinucleotide, Reduced Form, Disodium Salt, 50 mg for $22.60 __OR__ β-Nicotinamide adenine dinucleotide, reduced disodium salt, 15 vials with 5 mg per vial for $154.50 (4) a-Glycerophosphate Dehydrogenase/Triosephosphate Isomerase, 250 units at 75-200 units/mg for $48.40
 * Total reagent cost: $188.40 - $320.30 (depending on which reagent chosen for 3.)**


 * Structure Available (PDB or Homology model)**
 * -- PDB # or closest PDB entry if using homology model:** Direct match-[]
 * -- Query Coverage (if not direct match):** N/A
 * -- Max Ident (if not direct match):** N/A
 * Druggable Target (see Databases for this):** yes
 * Current Inhibitors:**
 * Expression Information (has it been expressed in bacterial cells):** expressed in E. coli
 * Purification Method:**
 * Image of protein (PyMol or etc):** see below
 * || [[image:http://www.pdb.org/pdb/images/1a5c_bio_r_500.jpg caption="http://www.pdb.org/pdb/images/1a5c_bio_r_500.jpg"]] ||
 * http://www.pdb.org/pdb/images/1a5c_bio_r_500.jpg ||  ||
 * [] ||

MAHCTEYMNAPKKLPADVAEELATTAQKLVQAGKGILAADESTQTIKKRFDNIKLENTIE NRASYRDLLFGTKGLGKFISGAILFEETLFQKNEAGVPMVNLLHNENIIPGIKVDKGLVN IPCTDEEKSTQGLDGLAERCKEYYKAGARFAKWRTVLVIDTAKGKPTDLSIHETAWGLAR YASICQQNRLVPIVEPEILADGPHSIEVCAVVTQKVLSCVFKALQENGVLLEGALLKPNM VTAGYECTAKTTTQDVGFLTVRTLRRTVPPALPGVVFLSGGQSEEEASVNLNSINALGPH PWALTFSYGRALQASVLNTWQGKKENVAKAREVLLQRAEANSLATYGKYKGGAGGENAGA SLYEKKYVY
 * Amino Acid Sequence:**


 * length of your protein in Amino Acids =** 369 AA
 * Molecular Weight of your protein in kiloDaltons using the [|Expasy ProtParam]website =** 40105.0 Daltons
 * Molar Extinction coefficient of your protein at 280 nm wavelength:**

Ext. coefficient = 40255 Abs 0.1% (=1 g/l) 1.004, assuming all pairs of Cys residues form cystines Ext. coefficient = 39880 Abs 0.1% (=1 g/l) 0.994, assuming all Cys residues are reduced


 * CDS Gene Sequence:**

(Cannot seem to find gene sequence for p. falciparum)


 * GC% Content for gene:**

ATGGCGCACTGCACTGAGTACATGAATGCGCCGAAGAAACTGCCGGCTGACGTTGCGGAA GAACTCGCGACCACTGCGCAAAAACTGGTTCAGGCGGGTAAAGGTATCCTGGCGGCTGAT GAATCTACCCAAACCATCAAGAAACGCTTCGACAACATCAAGCTGGAAAACACCATCGAA AACCGTGCGTCTTACCGCGACCTGCTGTTCGGCACCAAAGGTCTGGGTAAATTCATCTCT GGTGCGATCCTGTTCGAGGAGACGCTGTTCCAGAAAAACGAAGCGGGCGTTCCAATGGTT AATCTGCTGCACAACGAGAACATCATCCCGGGTATCAAAGTTGACAAAGGCCTCGTAAAT ATCCCGTGCACCGATGAGGAAAAATCTACGCAGGGTCTCGACGGTCTGGCTGAACGTTGC AAAGAATACTACAAAGCTGGTGCTCGCTTTGCTAAATGGCGTACCGTTCTGGTTATCGAC ACGGCGAAGGGCAAACCGACCGACCTGAGCATCCACGAAACCGCGTGGGGCCTCGCACGT TACGCGTCTATCTGCCAACAGAATCGTCTGGTTCCGATCGTTGAACCGGAAATTCTGGCG GACGGCCCTCACTCTATCGAAGTTTGCGCGGTTGTTACGCAGAAGGTTCTGTCTTGCGTA TTCAAAGCTCTGCAAGAAAATGGTGTGCTGCTGGAAGGTGCTCTGCTGAAACCGAACATG GTTACGGCGGGTTACGAATGTACTGCGAAGACGACCACCCAGGACGTTGGTTTCCTGACC GTTCGTACGCTGCGTCGTACGGTTCCTCCGGCGCTGCCGGGTGTTGTATTCCTGAGCGGT GGTCAGTCTGAGGAAGAAGCATCCGTTAACCTGAATTCTATCAATGCGCTCGGTCCGCAC CCGTGGGCGCTCACGTTCTCTTATGGTCGTGCGCTGCAGGCGTCTGTTCTGAACACCTGG CAGGGTAAAAAAGAAAACGTGGCGAAAGCGCGTGAAGTTCTCCTGCAACGCGCTGAAGCG AACTCTCTGGCGACCTACGGTAAGTATAAAGGCGGTGCGGGTGGTGAAAACGCTGGCGCG TCTCTGTACGAAAAGAAGTACGTTTACTAA
 * CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol:**


 * GC% Content for gene (codon optimized):** 53.7%


 * Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):**
 * link to DNA Works output text file : **[]

1 ATGGCGCACTGCACTGA 17 28 TTAGTAAACGTACTTCTTTTCGTACAGAGACGCGCCAGC 39
 * Primer design results for 'tail' primers (this is just 2 sequences):**


 * 08/31/2012:**

-Wolbachia, phosphatidylglycerophosphatase A (Pgp A) 3.1.3.27 -It is essential and not complex -unfortunately, it is a membrane protein -Try: plasmodium falciparum -glutathione reductase (too present in humans) -thioredoxin reductase or 3-oxoacyl-(acyl-carrier protein) reductase -thioredoxin reductase -human and bacteria forms are only ~20% similar - __serine/threonine protein phosphatase__ -http://www.promega.com/resources/protocols/technical-bulletins/0/serine-threonine-phosphatase-assay-system-protocol/ -http://www.pdb.org/pdb/explore/explore.do?structureId=1S95 -yes (at least 0.5) -http://www.bindingdb.org/bind/BySourceOrganism.jsp (serine/threonine protein phosphatase doesn't seem to be there) -http://www.malariajournal.com/content/1/1/5 -yes <span style="display: block; height: 1px; left: -40px; overflow: hidden; position: absolute; top: 7676.5px; width: 1px;">
 * 1. Choose organism**
 * 2. Assayable Enzyme Target**
 * 3. Crystal Structure**
 * 4. Druggable Target**
 * 5. Check current inhibitors**
 * 6. Expression**
 * 7. Gene Available**