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Team:UiOslo Norway/Notebook
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<div class="row"> | <div class="row"> | ||
<div class="col-md-12 borderTopRow person-box"> | <div class="col-md-12 borderTopRow person-box"> | ||
| - | <h3> | + | <h3>Friday</h3> |
<p>Miniprep of the T1 terminator O/N culture. 3 MP's made in parallel with NanoDrop DNA concentrations:</p> | <p>Miniprep of the T1 terminator O/N culture. 3 MP's made in parallel with NanoDrop DNA concentrations:</p> | ||
<ul> | <ul> | ||
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<div class="row"> | <div class="row"> | ||
<h1>Week 28</h1> | <h1>Week 28</h1> | ||
| + | </div> | ||
| + | <div class="row"> | ||
| + | <div class="col-md-12 borderTopRow person-box"> | ||
| + | <h3>Monday</h3> | ||
| + | <p>Batch of LB agar plates w/Ampicillin was made.</p> | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="row"> | ||
| + | <div class="col-md-12 borderTopRow person-box"> | ||
| + | <h3>Tuesday</h3> | ||
| + | <p>The results of the 3A assembly was poor, with either no growth at all or carpet growth.</p> | ||
| + | <p>The O/N cultures from the day before was made without using antibiotics, so the cultures was transferred to fresh medium with antibiotics to try to select for the right bacteria.</p> | ||
| + | <p>2I, 11N, 2K and 19E was plated on LB agar with 0.1% arabinose.</p> | ||
| + | <p>New batch of LB agar was made, using LB broth powder.</p> | ||
| + | </div> | ||
</div> | </div> | ||
<div class="row"> | <div class="row"> | ||
<div class="col-md-12 borderTopRow person-box"> | <div class="col-md-12 borderTopRow person-box"> | ||
<h3>Friday</h3> | <h3>Friday</h3> | ||
| - | <p> | + | <p>O/N cultures was miniprepped and freeze stocks were made of:</p> |
| + | <ul> | ||
| + | <li>pBad + 19E in pSB1A3</li> | ||
| + | <li>pBad + 2K in pSB1A3</li> | ||
| + | <li>pBad + 2I in pSB1A3</li> | ||
| + | <li>pBad + 11N in pSB1a3</li> | ||
| + | <li>13L in pSB1A2</li> | ||
| + | <li>His alpha stop in psB1A3</li> | ||
| + | <li>19E in pSB1C3</li> | ||
| + | </ul> | ||
| + | <p>All in TOP10 E.coli cells.</p> | ||
| + | <p>New transformation was set up using the following ligation products:</p> | ||
| + | <ul> | ||
| + | <li>LacZ alpha NS (from AB strain) + pBAD + pSB1A3</li> | ||
| + | <li>LacZ alpha NS (from BL strain) + pBAD + pSB1A3</li> | ||
| + | <li>LacZ beta NS (from AB strain) + pBAD + pSB1A3</li> | ||
| + | <li>LacZ alpha NS (from BL strain) + pBAD + pSB1A3</li> | ||
| + | </ul> | ||
| + | <p>The NCM17 E.coli strain was received from Yale E.coli Repository, plated and overnight cultured.</p> | ||
</div> | </div> | ||
</div> | </div> | ||
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<div class="row"> | <div class="row"> | ||
<div class="col-md-12 borderTopRow person-box"> | <div class="col-md-12 borderTopRow person-box"> | ||
| - | <h3> | + | <h3>Monday</h3> |
| - | <p> | + | <p>Freeze stock was made of NCM17 strain.</p> |
| + | </div> | ||
| + | </div> | ||
| + | <div class="row"> | ||
| + | <div class="col-md-12 borderTopRow person-box"> | ||
| + | <h3>Tuesday</h3> | ||
| + | <p>O/N culture of bacteria transformed with 17O was miniprepped.</p> | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="row"> | ||
| + | <div class="col-md-12 borderTopRow person-box"> | ||
| + | <h3>Wednesday</h3> | ||
| + | <p>Made 20 LB plates with kanamycin.</p> | ||
| + | <p>The miniprep of LacZ beta NS in pSB1C3 was run on a gel. The bond was the right size.</p> | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="row"> | ||
| + | <div class="col-md-12 borderTopRow person-box"> | ||
| + | <h3>Thursday</h3> | ||
| + | <p>The NCM17 strain was made competent (Using Jack Leo’s protocol) and put in freezer.</p> | ||
</div> | </div> | ||
</div> | </div> | ||
| Line 396: | Line 448: | ||
<div class="row"> | <div class="row"> | ||
<div class="col-md-12 borderTopRow person-box"> | <div class="col-md-12 borderTopRow person-box"> | ||
| - | <h3> | + | <h3>Monday</h3> |
| - | <p> | + | <p>The N-terminal His tag was made by mixing reverse and forward primers, putting the solution in a 96oC waterbath and left to cool.</p> |
| + | <p>Made O/N of TOP10 and NCM17.</p> | ||
| + | <p>Made O/N of double terminator in pSB1C3 (too much medium compared to bacteria - no growth).</p> | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="row"> | ||
| + | <div class="col-md-12 borderTopRow person-box"> | ||
| + | <h3>Tuesday</h3> | ||
| + | <p>O/N cultures of TOP10 and NCM17 was diluted and Jack’s protocol was followed in order to make them competent.</p> | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="row"> | ||
| + | <div class="col-md-12 borderTopRow person-box"> | ||
| + | <h3>Wednesday</h3> | ||
| + | <p>The digestion of the 3A assembly protocol was done with:</p> | ||
| + | <p>N-His tag, pBad and constitutive promoter (17O) as part A’s (cut with EcoRI and SpeI). LacZ alpha stop, lacZ beta stop and RBS as part B’s (cut with PstI and XbaI). pSB1A3 and pSB1C3 as plasmids (cut with PstI, EcoRI).</p> | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="row"> | ||
| + | <div class="col-md-12 borderTopRow person-box"> | ||
| + | <h3>Thursday</h3> | ||
| + | <p>Colonies from a plate with growth of LacZ-beta transformed bacteria were put in tubes, mixed with 20 µl water, heated to 96oC for 10 minutes and then centrifuged at 11.000 rpm for 10 minutes.</p> | ||
| + | <p>A PCR was set up in order to amplify the LacZ beta fragment, but there was no result on the gel.</p> | ||
</div> | </div> | ||
</div> | </div> | ||
Revision as of 09:57, 6 October 2014
Notebook
Welcome to our notebook! Here you can see what we've done each week in the lab, in media and everything else. Click below to navigate between weeks.
Week 18
Thursday
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- This
- And that
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Friday
Nothing much yo.
Week 19
Thursday
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- This
- And that
This text is in italics because of the em tag.
This text is bold because of the strong tag
Friday
Nothing much yo.
Week 20
Friday
Nothing much yo.
Week 21
Friday
Nothing much yo.
Week 22
Friday
Nothing much yo.
Week 23
Friday
Nothing much yo.
Week 24
Thursday William
PCR on BL21 and AB2848 strain on bacteria to retrieve LacZ-beta gene. Used LacZ-beta STOP primer.
Agarose gel electrophoresis result: Band about 3 kb for the BL21 strain. Probably LacZ-beta.
Friday William
PCR clean-up using Machery-Nagel PCR clean-up kit. Followed protocol from the kit.
PCR amplification of the cleaned DNA with and without stop codon.
Week 25
Tuesday
PCR clean-up using Machery-Nagel PCR clean-up kit on DNA with and without stop codon.
We did step 1 of making E. coli TOP10 cells competent (incubation of cells from freeze stock in LB medium with streptomycin.
We analyzed the DNA concentration using NanoDrop:
- LacZ-beta with stop codon: 230 ng/µl
- LacZ-beta without stop codon: 300 ng/µl
Wednesday
Finished Jack's protocol for making competent cells.
Attempted to transform bacteria with these parts:
- LacZ-alpha 22D (plate 2)
- 10X His-tag 5P (plate 3)
- pBad strong promoter 14A (plate 3)
- T1 terminator 1D (plate 1)
- Double terminator 3D (plate 1)
Parts were resuspended in accordance to iGEM protocol and for the transformation we used Jack's protocol.
Plates for E. coli growth were made and the transformation products were plated.
Thursday
Growth of 10X his-tag and double terminator transformed bacteria observed. Overnight (O/N) cultures were made of these.
LacZ-alpha, pBad and T1 were attempted transformed again.
New batch of LB-plates were made with chloramphenicol.
Friday
Restriction cutting of LacZ-beta STOP cleaned PCR product, LacZ-beta NONSTOP cleaned PCR-product and pSB1C3 were attempted using restriction enzymes XbaI and PstI. Incubation in accordance with iGEM restriction digest protocol. The tube with pSB1C3 was not mixed with water before pipetting.
The concentration of the digested products were measured in NanoDrop:
- LacZ-beta stop: 14.4 ng/µl
- LacZ-beta non stop: 23.3 ng/µl
- pSB1C3: 26.5 ng/µl
Ligation was attempted on the digestions using T4 DNA ligase and following the iGEM ligation protocol.
Freeze stocks of the O/N cultures of 10X His-tag and double terminator were made.
Minipreps using Promega Wizard Plus SV miniprep kit was attempted on saturated 10X his-tag and double terminator cultures.
Cultures observed on pBad, LacZ-alpha and T1-terminator plates. O/N cultures made during the weekend.
Week 26
Tuesday
No colonies seen on the LacZ-beta stop and non stop plates.
The digestion was repeated, but now with solubilized pSB1C3.
Thursday
Made O/N culture of T1 terminator from freeze stock in order to obtain pSB1C3 plasmids in case the pSB1C3 from the tube in the kit is defective.
We tested the restriction digest protocol by using a part from the kit we don't need (1A). Due to issues with the agarose gel we started over using another part (1K) that we left it in 37C for 4 hours and then on 4C overnight.
Friday
Miniprep of the T1 terminator O/N culture. 3 MP's made in parallel with NanoDrop DNA concentrations:
- T1-1: 15.6 ng/µl
- T1-2: 16.7 ng/µl
- T1-3: 16.2 ng/µl
The T1-1 was digested using XbaI and PstI following the iGEM digestion protocol.
The product was run on a gel and we cut out the pSB1C3 part using a scalpel and stored it in a eppendorf tube in the fridge.
Week 27
Monday
We used the NucleoSpin gel clean-up kit and protocol in order to isolate the XbaI/PstI-cut pSB1C3 from the gel piece. Concentration measured with NanoDrop: 10-15 ng/µl.
PCR was done on LacZ-beta with stop and non stop reverse primers using Q5 polymerase.
Thursday
O/N cultures were made of the colonies of transformed pSB1C3/LacZ-beta non-stop.
PCR was run with VR2 and VF primers for confirmation.
Friday
3A assembly was attempted with the bricks pBad and 2I, 2K, 19E and 11N, using pSB1A3.
Week 28
Monday
Batch of LB agar plates w/Ampicillin was made.
Tuesday
The results of the 3A assembly was poor, with either no growth at all or carpet growth.
The O/N cultures from the day before was made without using antibiotics, so the cultures was transferred to fresh medium with antibiotics to try to select for the right bacteria.
2I, 11N, 2K and 19E was plated on LB agar with 0.1% arabinose.
New batch of LB agar was made, using LB broth powder.
Friday
O/N cultures was miniprepped and freeze stocks were made of:
- pBad + 19E in pSB1A3
- pBad + 2K in pSB1A3
- pBad + 2I in pSB1A3
- pBad + 11N in pSB1a3
- 13L in pSB1A2
- His alpha stop in psB1A3
- 19E in pSB1C3
All in TOP10 E.coli cells.
New transformation was set up using the following ligation products:
- LacZ alpha NS (from AB strain) + pBAD + pSB1A3
- LacZ alpha NS (from BL strain) + pBAD + pSB1A3
- LacZ beta NS (from AB strain) + pBAD + pSB1A3
- LacZ alpha NS (from BL strain) + pBAD + pSB1A3
The NCM17 E.coli strain was received from Yale E.coli Repository, plated and overnight cultured.
Week 29
Monday
Freeze stock was made of NCM17 strain.
Tuesday
O/N culture of bacteria transformed with 17O was miniprepped.
Wednesday
Made 20 LB plates with kanamycin.
The miniprep of LacZ beta NS in pSB1C3 was run on a gel. The bond was the right size.
Thursday
The NCM17 strain was made competent (Using Jack Leo’s protocol) and put in freezer.
Week 30
Friday
Nothing much yo.
Week 31
Friday
Nothing much yo.
Week 32
Monday
The N-terminal His tag was made by mixing reverse and forward primers, putting the solution in a 96oC waterbath and left to cool.
Made O/N of TOP10 and NCM17.
Made O/N of double terminator in pSB1C3 (too much medium compared to bacteria - no growth).
Tuesday
O/N cultures of TOP10 and NCM17 was diluted and Jack’s protocol was followed in order to make them competent.
Wednesday
The digestion of the 3A assembly protocol was done with:
N-His tag, pBad and constitutive promoter (17O) as part A’s (cut with EcoRI and SpeI). LacZ alpha stop, lacZ beta stop and RBS as part B’s (cut with PstI and XbaI). pSB1A3 and pSB1C3 as plasmids (cut with PstI, EcoRI).
Thursday
Colonies from a plate with growth of LacZ-beta transformed bacteria were put in tubes, mixed with 20 µl water, heated to 96oC for 10 minutes and then centrifuged at 11.000 rpm for 10 minutes.
A PCR was set up in order to amplify the LacZ beta fragment, but there was no result on the gel.
Week 33
Friday
Nothing much yo.
Week 34
Friday
Nothing much yo.
Week 35
Friday
Nothing much yo.
Week 36
Friday
Nothing much yo.
Week 37
Friday
Nothing much yo.
Week 38
Friday
Nothing much yo.
Week 39
Friday
Nothing much yo.
Week 40
Friday
Nothing much yo.
Week 41
Friday
Nothing much yo.
Week 42
Friday
Nothing much yo.
Week 43
Friday
Nothing much yo.
Week 44
Friday
Nothing much yo.
