Team:EPF-Lausanne/Notebook/October2011

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Revision as of 09:19, 17 October 2011

Sunday, October 16 2011

Vincent came in to work on the blunt-end cloning for the inserts (T7 promoters + Lysis) and the psB3K1 vector. The idea is to use the T4 DNA polymerase, which has a 3' to 5' exonuclease, to fill-in and chew away the ends till we have blunt ends. I added the protocol to the appropriate protocol section. After running through this protocol, the T7 inserts had an average concentration of 40 ng/uL while the backbone vector K1 had a concentration of 90 ng/uL (to within Nanodrop measurement error). Those concentrations are high enough to allow us to move on to the next step which is phosphorylating the T7 inserts (not the backbone!) for ligation.

The phosphorylation step is needed because the ligation (via ligase enzyme) requires that one of the 5' termini have a phosphate residue that will induce phosphodiester bond. If both the insert and the backbone have the phosphate residue, then the backbone may bind to itself, leaving the insert out -- which would defeat the purpose of ligation.

The specifics of the blunt-end protocol were as follows:

For the T7 + Lysis inserts,

H20 - 11.25 uL
NEBuffer2 (10X, found in enzyme ice box) - 3 uL (needs to be at a final concentration of 1X)
dNTP (10 mM) - 0.3 (needs to be at a final concentration of 100 uM)
template - 15 uL (for 1.5 ug of DNA, assuming the T7 + Lysis was at a concentration of 100 ng/uL)
T4 Polymerase - 0.45 uL (it comes at a "concentration" of 3000 units/mL and we want 1.5 units)

I made a master mix of everything except the template DNA, which then went into separate PCR tubes. The total is a 30 uL reaction. The tubes were placed in the Bio-Rad machine were we setup a protocol (called BLUNTEND) that keeps the tubes at a constant 12 C for 15 minutes. If you want, you can add EDTA to a final concentration of 10 mM (as advised by New England Biolabs) and heat activate the samples at 75 C for 20 minutes. We decided that it was not necessary in our case.

For the K1 backbone, the recipe was slightly different due to the higher concentration of blunted DNA we wanted to achieve:

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-{{:Team:EPF-Lausanne/Templates/Header|title=Notebook: October 2011}}
+== Sunday, October 16 2011 ==
-== Wednesday, October 12 2011 ==
+Vincent came in to work on the blunt-end cloning for the inserts (T7 promoters + Lysis) and the psB3K1 vector. The idea is to use the T4 DNA polymerase, which has a 3' to 5' exonuclease, to fill-in and chew away the ends till we have blunt ends. I added the protocol to the appropriate protocol section. After running through this protocol, the T7 inserts had an average concentration of 40 ng/uL while the backbone vector K1 had a concentration of 90 ng/uL (to within Nanodrop measurement error). Those concentrations are high enough to allow us to move on to the next step which is phosphorylating the T7 inserts (not the backbone!) for ligation.
-Over the weekend, Matt kindly made gene-specific lysis (36 uL). With Vincent's help for finding a convenient Tm calculator for single-nucleotide mutants, he made the extension PCR to add the 12 T7 promoters ahead of the lysis cassette. Vincent ran the gel for them and the results (thanks to Matt's insane Keynote skillz) are found below.
+The phosphorylation step is needed because the ligation (via ligase enzyme) requires that one of the 5' termini have a phosphate residue that will induce phosphodiester bond. If both the insert and the backbone have the phosphate residue, then the backbone may bind to itself, leaving the insert out -- which would defeat the purpose of ligation.
-[[File:T7variants_step2.pdf|500px]]
+The specifics of the blunt-end protocol were as follows:
-Vdog made a PCR to amplify the pSB3K1 backbone. Just so he doesn't forget, here's the protocol:
+For the T7 + Lysis inserts,
-* 1 uL 10mM DNTP mix
+* H20 - 11.25 uL
-* 10 uL buffer with MgCl2 (part of the Roche pack)
+* NEBuffer2 (10X, found in enzyme ice box) - 3 uL (needs to be at a final concentration of 1X)
-* .5 uL Polymerase (Expand Hifi regular)
+* dNTP (10 mM) - 0.3 (needs to be at a final concentration of 100 uM)
-* .5 uL Template (psB3K1 1:10. found in t7 promoters box -- the ligated version is in the ? box)
+* template - 15 uL (for 1.5 ug of DNA, assuming the T7 + Lysis was at a concentration of 100 ng/uL)
-* 1 uL of each primer (50 uM)
+* T4 Polymerase - 0.45 uL (it comes at a "concentration" of 3000 units/mL and we want 1.5 units)
-* ddH20 to make 50 uL (36 uL if my count is right)
-The elongation temperature was 68 C (as indicated by Roche pamphlet) for 3 minutes (for a 3 kb piece). The annealing temp is 52 C (as stated in Alessandro's sheet in the Dropbox). Vdog ran a touchdown PCR from 62 to 52 C) using 3 50 uL reactions. If all goes well, we should have 150 uL of K1 ready to go. I will run the gel tomorrow.
+I made a master mix of everything except the template DNA, which then went into separate PCR tubes. The total is a 30 uL reaction. The tubes were placed in the Bio-Rad machine were we setup a protocol (called BLUNTEND) that keeps the tubes at a constant 12 C for 15 minutes. If you want, you can add EDTA to a final concentration of 10 mM (as advised by New England Biolabs) and heat activate the samples at 75 C for 20 minutes. We decided that it was not necessary in our case.
-== Thursday, October 13 2011 ==
+For the K1 backbone, the recipe was slightly different due to the higher concentration of blunted DNA we wanted to achieve:
-Vincent ran the gel from yesterday's PCR, and there were three nice bands at the 2744 bp level. Alessandro's sheet in the Dropbox had predicted that amount (I mistakingly thought it was around 3 kb because I looked it up on the registry but the part we are amplifying is considerably shorter than the full plasmid). The PCR purification of the T7+Lysis and the K1 backbone went down perfectly even though the purification kit was out of spin columns (I had to use those from a plasmid mini prep kit). The average concentration was 100 ng/uL for the promoter+lysis constructs and 130 ng/uL for the K1 backbone. In the elution step, I used 30 uL instead of the prescribed 50 uL, since we wanted high concentrations.
-The next step is to blunt the ends of both the plasmid backbone and the promoters+lysis constructs. For this, we used the T4 DNA polymerase from NEB and the associated protocol which I will add to the protocol section shortly (i.e. as soon as the TAs give me their approval). The technique seems straightforward but since the T4 polymerase has an exonuclease that reads from 3' to 5', the slightest mistake (i.e. overly elevated temperatures, excessive amounts of enzyme, not adding enough dNTPs, or letting the reaction run for too long) can all lead to recessed ends ... which would not be cool.
-Stay tuned.
-== Sunday, October 16 2011 ==
-Vincent came in to work on the blunt-end cloning for the inserts (T7 promoters + Lysis) and the psB3K1 vector. The idea is to use the T4 DNA polymerase, which has a 3' to 5' exonuclease, to fill-in and chew away the ends till we have blunt ends. I added the protocol to the appropriate protocol section.
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