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Team:Alberta/Notebook/Assembly Method
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<ul>Prepared acyl-NTPs by adding 50:u;L 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.</ul> | <ul>Prepared acyl-NTPs by adding 50:u;L 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.</ul> | ||
<ul>Set-up 4 tubes with 2:u;L of 0.5M EDTA. The EDTA will be used to inhibit Klenow Polymerase's enzyme activity.</ul> | <ul>Set-up 4 tubes with 2:u;L of 0.5M EDTA. The EDTA will be used to inhibit Klenow Polymerase's enzyme activity.</ul> | ||
| + | <ul>Prepare Blocking Solution by adding: 85:u;L cut pSB1C3 and AmpR, 4:u;L complete acyl-NTP mix and 10:u;L Klenow Buffer.</ul> | ||
| + | <ul>Transfer 20:u;L of Blocking Solution in one tube with 0.5M EDTA. This is the control of the experiment. It was placed in a 75 degrees Celsius water bath for 20 minutes to heat inactivate Klenow.</ul> | ||
| + | <ul>Add 5:u;L Klenow Polymerase to the remaining Blocking Solution and incubate at 37 degrees Celsius. | ||
| + | <ul>20:u;L of the Blocking Solution was transferred to the remaining tubes with 0.5M EDTA after 5, 10 and 20 minutes incubation at 37 degrees Celsius. These tubes were also heat inactivated at 75 degrees Celsius for 20 minutes. | ||
Revision as of 19:04, 23 July 2010
Contents |
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 200:u;l of Wash Buffer to beads and agitate. Centrifuge for 10 seconds and remove supernatant. Prewarm Elution Buffer in a 70 degrees Celsius 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 4:u;l 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 400:u;l 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 200:u;l 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
<p>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 / 60^2 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.
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:
- 28.3:u;L pSB1C3 with AmpR (176.5ng/:u;L nanodrop concentration)
- 60.7:u;L MilliQ H20
- 10.0:u;L 10X NEBuffer 4
- 1.0:u;L BsaI
- 100:u;L Total
Digestion #2 Protocol:
- 28.7:u;L pSB1C3 with AmpR (174.1ng/:u;L nanodrop concentration)
- 60.3:u;L MilliQ H20
- 10.0:u;L 10X NEBuffer 4
- 1.0:u;L BsaI
- 100:u;L Total
Both were incubated at 50 degrees Celsius for 1 hour, were PCR Purified using the Qiagen PCR Purification Kit and ran on a 1.0% agarose gel to check for completion.
<---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)
Protocol:
- Prepared acyl-NTPs by adding 50:u;L 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 2:u;L of 0.5M EDTA. The EDTA will be used to inhibit Klenow Polymerase's enzyme activity.
- Prepare Blocking Solution by adding: 85:u;L cut pSB1C3 and AmpR, 4:u;L complete acyl-NTP mix and 10:u;L Klenow Buffer.
- Transfer 20:u;L of Blocking Solution in one tube with 0.5M EDTA. This is the control of the experiment. It was placed in a 75 degrees Celsius water bath for 20 minutes to heat inactivate Klenow.
- Add 5:u;L Klenow Polymerase to the remaining Blocking Solution and incubate at 37 degrees Celsius.
- 20:u;L of the Blocking Solution was transferred to the remaining tubes with 0.5M EDTA after 5, 10 and 20 minutes incubation at 37 degrees Celsius. These tubes were also heat inactivated at 75 degrees Celsius for 20 minutes.
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