Difference between revisions of "Team:UCLA/Notebook/Spider Silk Genetics"
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<summary>7/12/2015 - 7/18/2015</summary> | <summary>7/12/2015 - 7/18/2015</summary> | ||
− | <a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/17_July_2015"><li>7/17/2015 - Miniprep, and | + | <a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/17_July_2015"><li>7/17/2015 - Miniprep, and Sequencing for M2-3(T7), M2-6(T7), and M2-9(1C3)</li></a> |
<a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/16_July_2015"><li>7/16/2015 - ICA for MaSp2 12-mer, Liquid Culture for MaSp2 3-mer(T7), MaSp2 6-mer(T7), and MaSp2 9-mer(pSB1C3)</li></a> | <a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/16_July_2015"><li>7/16/2015 - ICA for MaSp2 12-mer, Liquid Culture for MaSp2 3-mer(T7), MaSp2 6-mer(T7), and MaSp2 9-mer(pSB1C3)</li></a> | ||
<a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/15_July_2015"><li>7/15/2015 - Subcloning MaSp2 3-, 6-mer into T7 expression Vector, Cloning MaSp2 9-mer into pSB1C3</li></a> | <a href="https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/15_July_2015"><li>7/15/2015 - Subcloning MaSp2 3-, 6-mer into T7 expression Vector, Cloning MaSp2 9-mer into pSB1C3</li></a> |
Revision as of 15:57, 21 July 2015
Spider Silk Genetics Notebook
7/12/2015 - 7/18/2015
7/5/2015 - 7/11/2015
6/28/2015 - 7/4/2015
6/21/2015 - 6/27/2015
6/14/2015 - 6/20/2015
5/31/2015 - 6/6/2015
5/24/2015 - 5/30/2015
5/17/2015 - 5/23/2015
5/19/2015 - PCR amplification test for MaSp plasmid
5/19/2015
1 PCR Amplification of MaSp Plasmids
∙ Set up four 25 uL PCR reactions to test amplification. Used two pairs of primers and two different cycling conditions.
∙ Used MaSp2 2CA as template. Diluted 1:1000 to get 219 pg/uL. Created 2 master-mixes, one with each primer pair.
∙ Tested VF2/VR primers and post-elution primers using 17 cycles and 15 cycles using protocol below:
1x Reaction (uL) | 2x Reaction (uL) | |
5x Q5 Buffer | 5 | 10 |
10 mM dNTPs | 0.5 | 1 |
10 uM For | 1.25 | 2.5 |
10 uM Rev | 1.25 | 2.5 |
Template (110 pg) | 0.5 | 1 |
5x GC Buffer | 5 | 10 |
Q5 Polymerase | 0.25 | 0.5 |
ddH2O | 11.25 | 22.5 |
Total | 25 | 50 |
98 C | 30 sec |
98 C | 10 sec |
66 C | 15 sec |
72 C | 15 sec |
repeat from step 2 | 17x/15x |
72 C | 2 min |
12 C | hold |
∙ We used 66 C as annealing because that temperature worked for previous amplification with the post-elution primers. In addition, NEB Tm calculator indicates 66 C annealing for the VF2/VR primer pair.
2 Results
∙ Cast 2% TAE gel to visualize results. Used 2 uL of NEB 50 bp ladder.
∙ The VF/R primer pair amplifies our plasmid as expected, but the post-elution primers do not. The expected size for VF/R amplification is 443 bp.
∙ May be due to bad primers for post-elution. Re-order or remake? We need to find more of the post-elution primers.
5/10/2015 - 5/16/2015
5/13/2015 - NotI Concatemer Ligation
5/13/2015
1 NotI Concatemers
Used yesterday’s gel purified fragments. We performed ligation using a 5:1 ratio of insert to vector. Our vector size is 2046 bp. Our insert is 143 bp. For 1121 ng of vector, we need 391.75 ng of insert. We set up the ligation reaction as below:
Amount (uL) | |
Vector (112.12 ng/uL) | 10 |
Insert (52.63 ng/uL) | 10 |
10X T4 Ligase Buffer | 2.5 |
T4 Ligase | 3 |
Total | 25.5 |
We used excess insert because the recorded concentration from the nanodrop was higher than the theoretical yield. In addition, we used more T4 Ligase than in typical reactions due to the reaction having three times as many sticky ends to ligate.
2 Results
We cast a 0.8% TAE agarose gel to separate the bands. We ran this particular gel at 90 V for 2 hours. We excised four bands indicated for gel purification (Qiagen). The bottom most band may be due to vector self-ligation. The second band at 2.2 kb is probably singly ligated product.
In the future, we should CIP treat the vector so it doesn’t re-ligate.
5/12/2015 - T4/T7 Ligation Tests continued
5/12/2015
1 Gel Extraction
Performed gel extraction (Qiagen kit) of bands excised from yesterday’s gel. Results below:
Concentration (ng/uL) | A 260/280 | |
2AB | 41.5 | 4.47 |
2BC | 54.3 | 3.96 |
2CA | 17.6 | 3.21 |
2CA NotI ins. | 52.63 | 3.70 |
2CA NotI vec. | 112.12 | 2.50 |
2 2AB, 2BC, 2CA BsaI Ligation Test
Set up ligation tests for remaining conditions (see entry for 5/8/2015)for other tests. For each reaction, we used 50 ng of each DNA species.
2.1 T4 Ligase Reactions
2AB + 2BC | 2BC +2CA | |
10X T4 Ligase Buffer | 1.5 uL | 1.5 uL |
2AB (6.82 ng/uL) | 7.3 uL | 0 uL |
2BC (54.3 ng/uL) | 1 uL | 1 uL |
2CA (17.6 ng/uL) | 0 uL | 2.84 uL |
T4 Ligase | 1 uL | 1 uL |
ddH2O | 4.2 uL | 8.66 uL |
Total | 15 uL | 15 uL |
Reactions were incubated at 25 C for 10 min.
2.2 T7 Ligase Reactions
2CA + 2AB | 2BC + 2CA | |
2X T7 Ligase Buffer | 7.5 uL | 7.5 uL |
2AB | 1.2 uL | 0 uL |
2BC | 0 uL | 1 uL |
2CA | 2.84 uL | 2.84 uL |
T7 Ligase | 0.5 uL | 0.5 uL |
ddH2O | 2.96 uL | 3.16 uL |
Total | 15 uL | 15 uL |
Reactions were incubated at 25 C for 10 min.
3 Results
We cast a 2% TAE agarose gel to visualize the results. We used 2 uL of 50 bp ladder. See below.
5/11/2015 - BsaI Digestion of MaSp Plasmids
5/11/2015
1 2AB, 2BC, 2CA Ligation Test
1.1 BsaI digestion of 2AB, 2BC, 2CA
Used 5 ug of each DNA.
2AB (112 ng/uL) | 2BC (205 ng/uL) | 2CA (218.7 ng/uL) | |
BsaI | 4 uL | 4 uL | 4 uL |
Template | 44.6 uL | 24.39 uL | 22.86 uL |
Cutsmart | 5 uL | 5 uL | 5 uL |
ddH2O | 0 uL | 16.61 uL | 18.14 uL |
Total | 53.6 uL | 50 uL | 50 uL |
50 C | 2 hr | ||
65 C | 20 min |
We are conducting ligation tests between all possible combinations using T4 and T7 ligase
2 NotI Concatemers
Sri suggested using NotI to digest the plasmids and create a super plasmid that
contains multiple copies of the monomers.
Testing NotI digestion today using 2CA with the protocol below.
2CA (280 ng/uL) | |
NotI-HF | 2 uL |
DNA | 7.2 uL |
Cutsmart | 5 uL |
ddH2O | 35.8 uL |
Total | 50 uL |
37 C | 1 hr |
65 C | 20 min |
3 Results
Cast 200 mL of a 1.5% TAE agarose gel to visualize results of both BsaI and Not1 digestion.
5/3/2015 - 5/9/2015
5/8/2015 - T4/T7 Ligation Test
5/8/2015
1 T4/T7 Ligation Test
We tested the ability for the BsaI digested and gel purified monomers to ligate together. We ran these initial tests using both T4 and T7 DNA ligase using the following protocols (50 ng for each DNA species):
1.1 T4 Ligation
10x T4 Ligase Buffer | 1.5 uL | |
2AB | 4 uL | |
2CA | 5 uL | |
T4 Ligase | 1 uL | |
ddH2O | 3.5 uL | |
Total | 15 uL | |
25 C | 10 min | |
1.2 T7 Ligation
2x T7 Ligase Buffer | 7.5 uL | |
2AB | 4 uL | |
2BC | 5 uL | |
T7 Ligase | 0.5 uL | |
ddH2O | 0 uL | |
Total | 17 uL | |
25 C | 10 min | |
2 Results
We cast a 2% TAE gel to visualize the results. We use 2 uL of NEB 50 bp ladder. The gel was run at 150V for 35 min.
Our ligation test indicates that the new sticky ends exhibit specific binding. In both reactions, the ligation was incomplete due to the presence of a band at 100 bp. This may be due to non-equal amounts of DNA.
We plan to conduct this same test next week using all possible combinations between the three monomers to verify specific binding. We plan to test both T4 and T7.
5/7/2015 - BsaI Digestion of MaSp plasmids
5/7/2015
1 BsaI Digestion of 2AB, 2BC, 2CA
∙ Gel purification of yesterday’s BsaI digested fragments (Qiagen).
∙ Obtained approx. 7 ng/uL for each monomer. A 260/280 for all three were around 1.8. Will use these as is for ligation test tomorrow.
5/6/2015 - DNA Maxiprep
5/6/2015
∙ Spun down yesterday’s cells in 50 mL tubes for 15 min @ 6000x g @ 4 C.
∙ Used Maxiprep (Qiagen) kit to prepare DNA. Eluted in 400 uL EB. Concentrations from each are listed below:
Amount (ng/uL) | |
2AB | 112 |
2BC | 205 |
2CA | 219 |
∙ The DNA amounts are low. We probably need to improve our technique.
5/5/2015 - DNA Miniprep
5/5/2015
∙ Miniprep (Zymo) yesterday’s overnight bacterial culture. We also created glycerol stocks from these cultures.
Amount (ng/uL) | |
2AB | 380 |
2BC | 460 |
2CA | 219 |
∙ Prepared 100 mL culture of 2AB, 2BC, 2CA for Maxiprep tomorrow.
5/4/2015 - BsaI Digestion Tes
5/4/2015
1 BsaI Digestion Test for 2AB
Tested digestion of 5 ug of plasmid to see efficiency of digestion.
Amount (uL) | |
BsaI | 2 uL |
2AB (287 ng/uL) | 14.7 uL |
Cutsmart | 5 uL |
ddH2O | 28.28 |
Total | 50 uL |
50 C | 2 hr |
65 C | 20 min |
Results are shown below:
∙ The digestion process works, so we’ll be proceeding with this protocol.
∙ We gel extracted the digested insert band. The end result was 11.9 ng/uL, A (260/280): 4.4
2 BsaI Digestion for Post-elution primer amplified MaSp
We BsaI digested the PCR products from previous PCRs using the following protocol
2.1 BsaI Digest Protocol
2AB(61.43 ng/uL) | 2BC* | CA (56.65 ng/uL) | |
BsaI | 2 uL | 2 uL | 2 uL |
DNA (500 ng) | 8.14 uL | 18.01 uL | 8.83 uL |
10x Cutsmart | 5 uL | 5 uL | 5 uL |
ddH2O | 34.86 uL | 25 uL | 34.17 uL |
Total | 50 uL | 50 uL | 50 uL |
50 C | 2 hr | ||
65 C | 20 min |
*For 2BC, we combined 8.84 uL of 33.9 ng/uL solution and 9.17 uL and 33.9 ng/uL solution to get 500 ng total.
2.2 Purification
∙ PCR purified the post-elution PCR digestion products and got the following yields:
∙ Yield and absorbance for the digested inserts are not very good. This might be a problem due to the PCR process, even though product was generated.
3 Bacterial Propagation
∙ Grew 7 mL each of AB, BC, CA from glycerol stock for DNA extraction tomorrow.
4/26/2015 - 5/2/2015
5/1/2015 - Primer Exhaustion Calculation
5/1/2015
1 Calculation for Primer exhaustion
∙ Vector+insert = 2205 bp (150 bp insert) (2055 bp vector)
∙ 500 pg / 1431045 g/mol = 3.494 E -16 moles DNA
∙ 1.25 uL of 10 uM primers = 1.25 E -11 moles primers
∙ 1.25 E -11 / 3.494 E -16 = 35775.6
∙ log2(35775.6) = 15.1 cycles (assuming perfect PCR)
2 Modified PCR using post-elution primers
Redo PCR using same amounts of reagents, but with 20 cycles instead of 30. Made 2x 50 uL for each MaSp plasmid.
4/30/2015 - Post-elution PCR amplification of MaSp plasmids
4/30/2015
1 Post-elution primer PCR of MaSp plasmids
∙ The previous anomalous results may be due to a human factor. So we decided to have Olivia prepare the PCR today using the same protocol, but scaled to 3x 25 uL each for 2AB and 2BC. Results shown below:
4/29/2015 - Post-elution PCR amplification of MaSp plasmids
4/29/2015
1 Post-Elution primer PCR of MaSp 2 plasmids
∙ Performed same PCR reaction as yesterday, but with 2x 50 uL for each reaction. This was prepared as a master mix. Results shown below.
4/28/2015 - Post-elution PCR amplification of MaSp plasmids
4/28/2015
1 Post-Elution Primer PCR Amplification of MaSp2 Sequences
We used the post-elution primers to test amplify 2AB plasmid and to determine an appropriate annealing temperature. We used the following protocol:
1x Reaction (uL) | 4x Reaction (uL) | |
5x Q5 Buffer | 5 | 20 |
10 mM dNTPs | 0.5 | 2 |
10 uM For (F-03) | 1.25 | 5 |
10 uM Rev (G-03) | 1.25 | 5 |
2AB (500 pg) | 1.74 | 6.96 |
5x GC buffer | 5 | 20 |
Q5 Pol | 0.25 | 1 uL |
ddH2O | 10.01 | 40.04 |
Total | 25 | 100 |
98 C | 30 sec |
98 C | 10 sec |
69, 66.8, 65.4, 63 C | 15 sec |
72 C | 15 sec |
Repeat from step 2 | 30 x |
72 C | 2 min |
12 C | hold |
2 Results
∙ Base on these results, we decided to scale up to 3x 50 uL reactions for each monomer, and would use 66 C as the annealing temperature.
3 Amplification Again
We scaled up the above protocol for 3x 50 uL reactions for each monomer.
AB (287 pg/uL) | BC (260 pg/uL) | CA (223 pg/uL) | |
5x Q5 Buffer | 30 uL
| ||
10 mM dNTPs | 3 uL
| ||
10 uM For (F-03) | 7.5 uL
| ||
10 uM Rev (G-03) | 7.5 uL
| ||
Template (500 pg) | 5.22 uL | 5.76 uL | 6.73 uL |
Q5 Polymerase | 1.5 uL
| ||
5x GC enhancer | 30 uL
| ||
ddH2O | 65.28 uL | 64.74 uL | 63.77 uL |
Total | 150 uL |
98 C | 30 sec |
98 C | 10 sec |
66 C | 15 sec |
72 C | 15 sec |
repeat from step 2 | 30x |
72 C | 2 min |
12 C | hold |
4 Results for second PCR
∙ Visualized on 2% gel. Used 2 uL 50 bp ladder.
4/27/2015 - Initial ICA Ligation Test
4/27/2015
1 2AB, 2BC, 2CA Sticky End Ligation Test
AB+BC | BC+CA | CA+AB | AB+BC+CA | |
2x T7 Ligase Buffer | 5 uL | 5 uL | 5 uL | 5 uL |
ddH2O | 1.5 uL | 1.5 uL | 0.5 uL | 0 uL |
DNA 1 | 2 uL | 1 uL | 2 uL | 2 uL |
DNA 2 | 1 uL | 2 uL | 2 uL | 1 uL |
DNA 3 | n/a | n/a | n/a | 2 uL |
T7 Ligase | 0.5 uL | 0.5 uL | 0.5 ul | 0.5 uL |
Total | 10 uL | 10 uL | 10 uL | 10 uL |
25 C | 5 min |
65 C |
∙ The ligation test was run on 2% gel with 2 uL of 50 bp ladder. Results shown below.
4/19/2015 - 4/25/2015
4/24/2015 - Sending DNA for sequencing
4/24/2015
1 Sequencing of MaSp2 constructs
∙ Prepared each sample from yesterday for sequencing.
∙ Diluted 500 ng of each DNA species and 2.5 uL of VF2 primer into 15 uL in a PCR strip.
2 Results: 4/25/2015
∙ Used Clustal W2 to align sequencing results.
∙ Constructs 2AB-2, 2BC-1, and 2CA-2 are sequenced properly.
4/23/2015 - Miniprep DNA for sequencing
4/23/2015
1 Miniprep
∙ Prepared Glycerol stocks of each culture: 500 uL of bacteria + 500 uL 50% v/v glycerol.
∙ Used zymo miniprep kit to extract DNA from yesterday’s cells.
Concentration (ng/uL) | |
2AB-1 | 640 |
2AB-2 | 287 |
2BC-1 | 261 |
2BC-2 | 275 |
2CA-1 | 469 |
2CA-2 | 223 |
4/22/2015 - Colony PCR
4/22/2015
1 Bacterial Transformation
∙ Observed many colonies growing on the plates. We will do colony PCR to verify proper insertion.
2 Colony PCR
∙ Picked four colonies from each plate. Resuspended each each colony in 100 uL water. Used protocol below:
1x Reaction (uL) | 12x Reaction (uL) | |
5x Q5 buffer | 5 | 60 |
10 mM dNTPs | 0.5 | 6 |
10 uM For (VF2) | 1.25 | 15 |
10 uM Rev (post-elution G-03) | 1.25 | 15 |
Colony (Resuspended) | 1 uL | 1 uL |
5x GC Enhancer | 5 | 60 |
Q5 polymerase | 0.25 | 3 |
ddH2O | 10.75 | 129 |
Total | 25 | 300 |
98 C | 3 min |
98 C | 10 sec |
66 C | 15 sec |
72 C | 15 sec |
repeat from step 2 | 25 x |
72 C | 2 min |
12 C | hold |
3 Results
∙ We picked colonies 3 and 4 from each construct and grew 5 mL liquid culture for miniprep and sequencing tomorrow. Renumbered the cultures to 1 and 2 respectively.
4 Column purification of MaSp2 PCA
∙ Used zymo kit to purify yesterday’s MaSp2 PCA reactions. Eluted in 10 uL EB. Results below:
Concentration (ng/uL) | |
2AB | 279.34 |
2BC | 154.57 |
2CA | 236.36 |
5 BsaI Digestion of PCA products from 4/21/2015
∙ Set up 2x 25 uL reactions for each:
2AB(279 ng/uL) | 2BC (155 ng/uL) | 2CA (237 ng/uL) | |
DNA | 2 uL ( 560 ng) | 5 uL ( 770 ng) | 2.5 uL ( 590 ng) |
10x Cutsmart | 2.5 uL: | 2.5 uL | 2.5 uL |
ddH2O | 18.5 uL | 15.5 uL: | 18 uL |
BsaI | 2 uL | 2 uL | 2 uL |
Total | 25 uL | 25 uL | 25 uL |
50 C | 2 hr |
65 C | 20 min |
∙ The digestion products were column purified using zymo kit with the following yields:
Concentration (ng/uL) | A 260/280 | |
2AB | 24.7 | 2.09 |
2BC | 55.17 | 1.9 |
2CA | 22.38 | 2.14 |
4/21/2015 - Ligation of MaSp sequences into pSB1C3 and Transformation
4/21/2015
1 Ligation of MaSp 2AB, 2BC, 2CA in to pSB1C3
∙ Used ligation calculator to determine amounts of each insert to use to ligate into 50 ng of vector for a 3:1 ratio of insert to vector.
∙ 2000 bp vector, 170 bp insert.
∙ Determined 12.75 ng of insert to 50 ng vector. Proceeded with ligation using protocol below:
2AB (43.28 ng/uL) | 2BC (34.56 ng/uL) | 2CA (34.9 ng/uL) | |
T4 Ligase | 1 uL | 1 uL | 1 uL |
Insert (12.75 ng) | 0.30 uL | 0.370 uL | 0.365 uL |
Vector (50 ng) | 1.484 uL | 1.484 uL | 1.484 uL |
10x T4 Ligase Buffer | 2 uL | 2 uL | 2 uL |
ddH2O | 15.22 uL | 15.15 uL | 15.16 uL |
Total | 20 uL | 20 uL | 20 uL |
25 C | 20 min |
65 C | 10 min |
2 Bacterial Transformation
∙ Used 2 uL of each reaction to transform 50 uL competent cells.
∙ Add 200 uL of pre-warmed SOC to each tube.
∙ Plate 50 uL onto chloramphenicol plates. Incubate at 37 C overnight.
3 MaSp2 PCA
∙ Set up 2x 50 uL reactions for AB, BC, CA each using protocol from 4/13/2015 scaled to 50 uL with the following modifications:
4/20/2015 - PCR amplification of pSB1C3 and EcoRI, PstI digestion
4/20/2015
1 PCR Amplification of pSB1C3
∙ Due to poor amplification of pSB1C3 from friday, we are doing a temperature test today.
∙ Followed same protocol as 4/17/2015, but scaled to 3x 25 uL reactions.
∙ Decided to take nanodrop reading of the template Julian provided. It was 230 ng/uL. Instead of using stock concentration of template, we will use a 1:100 dilution. Results shown below.
∙ We gel extracted the bands together. 26.4 ng/uL, A 260/280: 1.56.
2 EcoRI and PstI Digestion of MaSp 2AB, 2BC, 2CA, and pSB1C3
∙ We pooled together all the linearized pSB1C3 that we had. Final concentration: 16.5 ng/uL.
Concentration (ng/uL) | |
2AB | 37.58 |
2BC | 42.8 |
2CA | 79.11 |
pSB1C3 | 16.5 |
2AB | 2BC | 2CA | pSB1C3 | |
EcoRI | 1 uL | 1 uL | 1 uL | 1 uL |
PstI | 1 uL | 1 uL | 1 uL | 1uL |
10x NEbuffer 2.1 | 5 uL | 5 uL | 5 uL | 5 uL |
DNA (500 ng) | 16 uL | 11 uL | 6 uL | 19 uL |
ddH2O | 27 uL | 32 uL | 37 uL | 24 uL |
Total | 50 uL | 50 uL | 50 uL | 50 uL |
37 C | 1 hr |
65 C | 20 min |
12 C | hold |
∙ Column purified all DNA after digestion using Zymo kit. Eluted in 10 uL of EB. Results shown below:
Concentration (ng/uL) | A 260/280 | |
2AB | 43.28 | 1.9 |
2BC | 34.56 | 1.7 |
2CA | 34.9 | 1.7 |
pSB1C3 | 33.7 | 1.61 |
4/12/2015 - 4/18/2015
4/17/2015 - Amplification of pSB1C3
4/17/2015
1 PCR amplification of pSB1C3
We set up a 50 uL reaction using the following protocol from Julian’s notebook.
Volume (uL) | |
5x Q5 Buffer | 10 |
10 mM dNTPs | 1 |
10 uM For (SB-prep 2EA) | 2.5 |
10 uM Rev (SB-prep 3P1) | 2.5 |
Template (Spycatcher from Julian) | 1 |
Q5 Polymerase | .0.5 |
ddH2O | 32.5 |
Total | 50 |
98 C | 30 sec |
98 C | 10 sec |
66 C | 20 sec |
72 C | 30 sec |
repeat from step 2 | 30 x |
72 C | 2 min |
12 C | hold |
2 Results
∙ Gel extraction (Qiagen) yielded 36.47 ng/uL, A 260/280: 1.91.
4/16/2015 - PCA of MaSp2 Pieces
4/16/2015
1 Polymerase Chain Assembly of MaSp 2AB sequence.
Repeated PCA of 2AB using protocol from 4/13/2015 with the follow modifications.
∙ We cast a 1.5% gel to visualize the results shown below.
2 Gel Extraction
Fasih gel extracted the two 2AB bands.
Amount (ng/uL) | A 260/280 | |
2AB-1 | 46.28 | 2.41 |
2AB-2 | 24.97 | 4.29 |
4/15/2015 - PCA of MaSp2 Pieces
4/15/2015
1 MaSp2 Polymerase Chain Assembly
Set up two 50 uL PCR reactions each for 2BC and 2CA using the same guidelines on 4/13/2015 with the following modifications:
2 Gel Purification of MaSp2 Monomers
∙ Visualized with 1.5% agarose gel. Similar result to yesterday’s gel.
∙ Used gel purification kit (zymo) to extract DNA from 2AB, 2BC, and 2CA.
Amount (ng/uL) | A 260/280 | |
2AB | 12.73 | 2.01 |
2BC | 42.8 | 1.93 |
2CA | 79.11 | 1.91 |
4/14/2015 - PCA of MaSp2 Pieces
4/14/2015
1 MaSp2 Polymerase Chain Assembly
Set up 2 50 uL reactions following the same protocol from yesterday scaled to 50 uL for 2AB. We included the following modifications:
2 Results
Cast 1.5% TAE agarose gel. Used 3 uL of NEB 50 bp ladder.
This protocol works better than the one previously. The target product at 170 bp is clearly more intense. While there is still some primer dimer formation, we will stick with this protocol.
4/13/2015 - PCA of MaSp2 Pieces
4/13/2015
1 Polymerase Chain Assembly
The primers corresponding to the new MaSp2 sequences with a valine added to the end came today. We conducted polymerase chain assembly to place the biobrick prefix/suffix and the BsaI restriction enzyme recognition sites with the designed sticky ends.
We used NEB’s Q5 PCR annealing temperature calculator to determine that an annealing temperature of 65 C was optimal. We also decided to test annealing at 61.4 C and 59 C. In addition, we planned to use the GC enhancer because our sequence is GC-rich. We prepared reactions as follows:
1x (uL) | 3x (uL) | |
5x Q5 Buffer | 5 uL | 15 uL |
10 mM dNTPs | 0.5 uL | 1.5 uL |
10 uM For | 1.25 uL | 3.75 uL |
10 uM Rev | 1.25 uL | 3.75 uL |
MaSp core (2.7 pg/uL) | 3.7 uL | 11.1 uL |
5x GC enhancer | 5 uL | 15 uL |
Q5 Polymerase | 0.25 uL | 0.75 uL |
ddH2O | 8.05 uL | 24.15 uL |
Total | 25 uL | 75 uL |
98 C | 30 s | |
98 C | 10 s | |
65/61.4/59 C | 15 s | |
72 C | 15 s | |
Repeat from step 2 | 20 x | |
72 C | 2 min | |
12 C | hold |
2 Results
We cast a 1.5% TAE gel to visualize the results. We used 2 uL of NEB 50 bp ladder.
Our results indicate that 65 degrees is too high, and does not have good yield, but 61.4 is too low and has non-specific amplification as shown by the smears. The band at 50 bp is attributed to primer dimer formation.
Based on our results, tomorrow, we will perform polymerase chain assembly with annealing at 64 C and with 25 cycles.
Contents
Goals
Our goal is to be able to implement Iterative Capped Assmebly (ICA) for use in designing custom spider silk genes. The spider silk protein has a highly repetitive primary structure, and its properties can be changed by changing the sequence and identities of these repeats. We want to be able to fully control the primary sequence of our silk gene, and develop a quick, efficient method of customizing assembly of the spider-silk genes.
Achievements
- We started testing ICA on 6/19/2015
- We successfully constructed biobricks that contain our silk genes and the sticky overhangs. 4/24/2015
- We proved that our designed sticky ends work as expected, and exhibit specific binding, showing that ICA can work in spider silk genes. 5/8/2015 and 5/12/2015
- We have established a protocol generating fragments to be used in ICA.
- We successfully characterized the use of ICA oligos with MaSp AB, BC, CA and showed that they could work in ICA! 6/3/2015
- We successfully made a 3-mer using the ICA protocol on M-270 Streptavidin Beads! 7/7/2015
- Find a list of our designed protocols here!
List of Parts
- [http://parts.igem.org/Part:BBa_K1763002 MaSp2 2AB]
- [http://parts.igem.org/Part:BBa_K1763003 MaSp2 2BC]
- [http://parts.igem.org/Part:BBa_K1763004 MaSp2 2CA]
What to accomplish next
We are working on optimizing PCR amplification of MaSp and BsaI digestion so as to get the most amount of working monomers for ICA.--DONE! see hereWe need to characterize our MaSp monomers and how they behave when used with the initiators, terminators, and capping oligos that are also used in ICA.--DONE! 6/3/2015We are going to start actual ICA soon.--STARTED! see 6/19/2015- We are optimizing ICA to get it to work just right!
- Our final goal is to make a several different 15-mers that have different rations of MaSp1 and MaSp2 that we can express and characterize.
Raw lab notebook entries
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