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From 2010.igem.org

Contents 1 25-05-2010 2 26-05-2010 3 27-05-2010 4 07-06-2010 5 08-06-2010 6 09-06-2010 7 10-06-2010 8 11-06-2010 9 14-06-2010 10 15-06-2010 11 16-06-2010 12 16-07-2010 13 19-07-2010 14 20-07-2010 15 21-07-2010

25-05-2010

Discussed an assembly method. Will start with a polyA tail Anchor oligo which will connect to a polyT tail which is bound to the bead. The anchor will consist of a primer region, a BsaI cut site, a buffer region, and finally, an A prime or B prime end. A byte with an appropriate end can be added to this anchor, and another byte can be added onto that, etc... The last thing to be added to the construct is a Terminator byte consisting of a buffer region, bsaI cut site, primer region and a polyT tail. This second primer sequence must be as uncomplimentary as possible to the anchor's primer site so as to minimize primers binding to each other when PCRing the assembled construct. The primers melting point must be around 65 C. After the construct is assembled on the bead, it is heated off and the polyA tail of the anchor binds to the polyT tail of the terminator and a plasmid is born.

We will purchase NEB oligo dT magnetic and cellulose beads which have a polyT tail (5' to 3') 25 nucleotides long. They are meant for mRNA isolation, but should work fine for our purposes. Used the IDT Analyzer software to determine what length of polyA tail is required for our anchor to have a melting temperature of 30 degrees Celcius (decided to change this melting temp later). This melting temp is sensitive to Na+ and Mg++ concentrations, so referred to ligase buffer and elution buffer for these concentrations.

The BsaI A end should look like this:

Designed an anchor with a polyA10 tail with a primer (Tm=65.1 C) region and the BsaI cut site with an A' end (ACCC).

26-05-2010

Designed new anchors, ordered oligos. Designed new anchors with larger polyA tail (and without hairpins, or self dimerization).

27-05-2010

No binding capacity specified in the manual for the beads in terms of mols or mass of DNA or beads. Binding capacity is given in terms of cells (because the beads are meant for mRNA isolation from cells). Will have to quantify binding capacity later.

Derivation of mole matching equation: ssDNA is ~330 grams/(mol*bp) moles = [concentration X volume] / [(330grams/mol X bp) X length] moles1 = moles2 c1v1/[(330g/mol X bp) X l1] = c2v2/[(330g/mol X bp) X l2] v1 = v2(c2 X l1/ c1 X l2)

Ordered new primers for anchor: GCG CGC CCG GTC TCA TGG GTC ACC CTC CC GGG AGG GTG ACC CAT GAG ACC GGC GCG C[A]12

07-06-2010

Made Wash Buffer, Elution Buffer and Low Salt Buffer for the mRNA Isolation Kit.

08-06-2010

Produce a modified protocol for Anchor binding to cellulose beads.

Protocol Preparation:

Allow everything to come to room temperature.

Spin 2000 to 5000g of beads in microcentrifuge for 10 seconds.

Remove supernatant without disturbing the beads.

Add 200μl of Wash Buffer to beads and agitate.

Centrifuge for 10 seconds and remove supernatant.

Prewarm Elution Buffer in a 70^oC water bath.

Isolation Procedure:

Apply DNA Anchor to cellulose beads, agitate and let it stand at room temperature for 5 minutes.

Microcentrifuge for 10 seconds.

Pipette supernatant back into original microcentrifuge.

[Add 4μl Wash Buffer to beads and agitate to resuspend. Transfer beads and wash buffer to column reservoir of spin column. Let it stand at room temperature for 2 minutes while agitating. Microcentrifuge for 10 seconds.] X3

Add 400μl Low Salt Buffer, resuspend by agitation for 2 seconds and microcentrifuge for 10 seconds.

Transfer and place spin column reservoir in a clean microcentrifuge tube.

[Add 200μl pre-warmed Elution Buffer to column reservoir.Agitate to resuspend beads and let it stand for 2 minutes.Microcentrifuge for 10 seconds] X2

Place eluent on ice.

09-06-2010

Calculated the mass of beads to determine the range of mg RNA could be isolated. 0.06g beads = 0.0599g; therefore, 0.06g can isolated 0.1 to 1 mg RNA.

Calculated the minimal and maximal rpm for 2000 to 5000g of beads using the following formula: a = 4(pi)²r(rpm)² / 60² So, when centrifuging the beads, the rpm must stay between 4700 to 7400 rpm. In the modified mRNA procedure, the centrifuge is set to 5500 rpm.

10-06-2010

Attempted to find binding capacity (in moles per mass of beads) by binding known masses of anchor to the bead, following the protocol, then qantifying the mass of DNA recovered off of the beads after eluting the DNA off of the bead. The cellulose beads settle very quickly without agitation. Measuring precise volumes of the beads was done by pipetting the cellulose bead slush into pcr tubes that were marked at 16 uL. To quantify the masses of DNA that: did not bind to the bead, came through the washes, and were eluted from the beads, the nano-drop 1000 spectrophotometer was used. The results showed that most of the anchor did not bind to the cellulose beads, and a very small amount of DNA was bound and eluted off of the beads. Possible reasons could be that the anchor was not binding, or there was too much anchor relative to the beads. A gel was also done. It showed that there was a lot of anchor that did not bind, and no anchor band was visible in the elution lane (which was expected based on the nanodrop reading).

11-06-2010

Repeated yesterday's experiment, but with a much larger amount of DNA. 1500 ng of anchor DNA to 16 uL of cellulose bead slush. There were no significant nanodrop readings except for the measurement of the amount of DNA that did not bind to the beads. Based on the nanodrop reading, over 1100 ng of DNA did not bind to the beads. The rest must have been washed away in the wash. Perhaps the melting temperature of the polyA12 tail is too low. We'll order one with a polyA18 tail and repeat the experiment.

14-06-2010

-Attempted to bind various masses of anchor to 16 uL of beads. Used 500, 1000, and 1500 ng of beads. Followed the modified mRNA isolation protocol created last week. Based on nanodrop readings, we recovered 560 ng from 1500 ng of initial DNA, 63 ng of 1000 ng of initial DNA, and 244 ng of 500 ng of DNA. Strange result of the 1000 ng of DNA, but a good sign that something appears to be binding to the cellulose beads. -To attempt to find the amount of anchor that will saturate the beads binding capacity, we used 1, 1.5, 2, and 2.5 ug of anchor and attempted to bind them to 16 uL of cellulose beads. Nanodrop readings multiplied by volume gave 600, 763, 1102, and 1730 ng of DNA not binding to the bead. What bound to the bead was 248, 1935, 4032, and 2470 ng of DNA. Obviously, something has caused the DNA readings to imply that more DNA was recovered than was put in initially. Later we found out that the readings are extremely sensitive to the small amount of residual washing solutions left over, causing the blanking process of the nanodrop to not accurately represent what solution the DNA was in.

15-06-2010

-The cellulose mRNA isolation kit came with a small amount of columns enough for only 8 trials. More were ordered. Will use magnetic beads in the meantime. -Created modified magnetic bead protocol based on the mRNA isolation protocol. Enough beads present in kit for 100 trials. -Magnetic Bead Trial #1: attempt to bind 100, 500, 1000, and 2500 ng of polyA12 anchor to 20 uL of polyT magnetic beads. Followed the modified magnetic bead protocol and eluted in 50 uL of elution buffer in a 50 C water bath. When nanodropping, apply magnet in case a small amount of magnetic particles were caught in the elution. Results: 80, 250, 305, and 460 ng of DNA were recovered from the beads after applying 100, 500, 1000 and 2500 ng of DNA initially. The magnetic beads appear to be more efficient at lower masses of DNA being bound. This appears encouraging, however, the nanodrop readings multiplied by the solution volume for the amount of DNA which did not bind initially were 335, 785, 970, and 2140 ng of DNA. The amount of DNA which did not bind and the amount that did does not add up to the amount of DNA added. This could be due to the residual solution left behind after washes. Will redo the experiment with less concentrated anchor and careful washes.

16-06-2010

-Magnetic Bead Trial 2: attempt to bind 500, 1000, 2500, and 5000 ng of anchor to 20 uL of beads. The mass of the DNA which didn't bind exceeded the mass of what was intitially mixed with the beads. This is obviously an error in measurement (caused by what!). The amount of DNA recovered was 0, 90, 70, and 250 ng of DNA. Looks like nothing bound to the beads. Possible cause of no DNA binding to bead: very low concentration of anchor meant a large reaction volume which would lead to a higher reaction time. Next time, use higher concentration anchor and increase binding time to an hour rather than 15 minutes (the mRNA isolation procedure calls for only 10 minutes).

16-07-2010

Solution Phase Assembly (Preparations)

Digested 10:u;g of pSB1C3 with Amp Resistance using BsaI. This digested DNA will be used to test: 1) whether acyl-NTPs will specifically incorporate to the cut ends of DNA by Klenow Polymerase and 2) if the acyl-NTPs block the ligation reaction between the vector (pSB1C3) and the insert (Amp Resistance). Two different minipreps of pSB1C3 with AmpR were used for the digestions.

Digestion #1 Protocol:

28.3μL pSB1C3 with AmpR (176.5ng/μL nanodrop concentration)

60.7μL MilliQ H₂0

10.0μL 10X NEBuffer 4

1.0μL BsaI

100μL Total

Digestion #2 Protocol:

28.7μL pSB1C3 with AmpR (174.1ng/μL nanodrop concentration)

60.3μL MilliQ H₂0

10.0μL 10X NEBuffer 4

1.0μL BsaI

100μL Total

Both were incubated at 50^oC for 1 hour, were PCR Purified using the Qiagen PCR Purification Kit and ran on a 1.0% agarose gel to check for completion.

Gel Electrophoresis:

<---16.07.10 Anh--->

Only lanes 6 to 8 were used. Lane 6 contains the kb+ ladder, while Lanes 7 and 8 contains the digested and purified pSB1C3 with AmpR fragments. Overall, the digestion went to completion.

19-07-2010

Solution Phase Assembly (Pre-test #1)

Used the digested pSB1C3 with AmpR that were prepared on 16-07-2010.

Protocol:

Prepared acyl-NTPs by adding 50μL of MilliQ H₂0 to each powdered acyl-NTP (acyl-ATP, acyl-CTP, acyl-GTP and acyl-TTP). Then, add equal volume amounts of each acyl-NTP solution in an eppendorf tube to produce the complete mix to be used in the pre-test.

Set-up 4 tubes with 2μL of 0.5M EDTA. The EDTA will be used to inhibit Klenow Polymerase's enzyme activity.

Prepare Blocking Solution by adding: 85μL cut pSB1C3 and AmpR, 4μL complete acyl-NTP mix and 10μL Klenow Buffer.

Transfer 20μL of Blocking Solution in one tube with 0.5M EDTA. This is the control of the experiment. It was placed in a 75^oC water bath for 20 minutes to heat inactivate Klenow.

Add 5μL Klenow Polymerase to the remaining Blocking Solution and incubate at 37^oC.

20μL of the Blocking Solution was transferred to the remaining tubes with 0.5M EDTA after 5, 10 and 20 minutes incubation at 37^oC. These tubes were also heat inactivated at 75^oC for 20 minutes.

All tubes were PCR Purified using the Qiagen PCR Purification Kit.

Ligation of digested pSB1C3 and AmpR was done after purification.
16μL digested pSB1C3 and AmpR with acyl-NTP.
2μL of 5X ligase buffer
2μL of T4 ligase

Ligations were incubated at room temperature for 1 hour.

20-07-2010

Solution Phase Assembly (Pre-test #1)

Ran the samples done on 19-07-2010 on a 1.0% agarose gel.

Gel Electrophoresis:

<---20.07.10 Anh--->

Lane 1 is the kb+ ladder. There are two controls: Lane 2 contains the cut pSB1C3 and Amp Resistance, and Lane 3 contains Blocking Solution but no Klenow Polymerase. Lanes 4 to 6 contains Blocking Solution with Klenow Polymerase but vary in incubation times for Klenow's enzyme reactivity. Lane 4 incubated for 5 minutes, Lane 5 incubated for 10 minutes and Lane 6 incubated for 20 minutes. Results: Lane 3 proceeded to ligate the DNA while Lanes 4 to 6 did not. Therefore, 5 minutes is the minimum amount of time to allow Klenow Polymerase to incorporate acyl-NTPs at the ends of digested pSB1C3 and AmpR, which prevents ligase's function to ligate the DNA.

21-07-2010

Solution Phase Assembly (Pre-test #2)

The next objective is to see if: 1) Klenow Polymerase can function efficiently in ligase buffer instead of Klenow Buffer to see if the PCR Purification step can be skipped, 2) to check whether acyl-NTPs are the one responsible for inhibiting ligase's activity, and 3) does 1X SDS enhance the visual of gel bands in 1.0% agarose gels.

Digestion of Kan Bsa B/A is needed before doing the experiment.

Digestion Protocol:

19.3μL Kan B/A Bsa (258.7ng/μL nanodrop concentration)

2.5μL 10X NEBuffer 4

2.2μL MilliQ H₂0

1.0μL Bsa I

<p>Incubate at 50^oC for 1 hour and PCR Clean-Up using Qiagen PCR Purification Kit.

Using the digested Kan Bsa B/A, proceed with the following protocols for Solution Phase Assembly (Pre-test #2): (Note that each tube contains a different solution mix with a total of 21μL, but they all have the same incubation times)

Protocol (Tube1):

16μL Kan B/A Bsa

2μL ligase buffer

2μL MilliQ H₂O

Incubate at 37^oC for 10 minutes.

Add 1μL MilliQ H₂O.

Incubate at room temperature for 1 hour.

Protocol (Tube2):

16μL Kan B/A Bsa

2μL ligase buffer

1μL complete acyl-NTP mix

1μL MilliQ H₂O

Incubate at 37^oC for 10 minutes.

Add 1μL T4 ligase.

Incubate at room temperature for 1 hour.

Protocol (Tube3):

16μL Kan B/A Bsa

2μL ligase buffer

1μL Klenow Polymerase

1μL MilliQ H₂O

Incubate at 37^oC for 10 minutes.

Add 1μL T4 ligase.

Incubate at room temperature for 1 hour.

Protocol (Tube4):

16μL Kan B/A Bsa

2μL ligase buffer

2μL MilliQ H₂O

Incubate at 37^oC for 10 minutes.

Add 1μL T4 ligase.

Incubate at room temperature for 1 hour.

Protocol (Tube5):

16μL Kan B/A Bsa

2μL ligase buffer

1μL complete acyl-NTP mix

1μL Klenow Polymerase

Incubate at 37^oC for 10 minutes.

Add 1μL T4 ligase.

Incubate at room temperature for 1 hour.

Gel Electrophorsis:

<---22.07.10 Anh--->

Lane 1 and 2 contain the kb+ ladder and the cut Kan B/A Bsa control respectively. The following sequential lanes are: Tube 1, 2, 3, 4, 5, and Tube 1, 2, 3, 4, 5 with 1.0% SDS added with the loading dye. Results: Add later.

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