Team:Macquarie Australia/Notebook3
From 2010.igem.org
Line 274: | Line 274: | ||
+ | <li type="disc">The reaction mastermix for the restriction digest to be performed on the Invitrogen Midi Prep kit purified plasmid was set up as per the following recipe (per sample): </li> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th> | ||
+ | Mastermix:</th> <th>Amount per sample (uL)</th></tr> | ||
+ | <tr> | ||
+ | <td> Gibco H2O</td> <td> 2.5</td> </tr> | ||
+ | <tr> | ||
+ | <td> 10x Buffer</td> <td>6.0</td></tr> | ||
+ | <tr> | ||
+ | <td> BSA (10ug/uL)</td> <td> 0.5</td></tr> | ||
+ | <tr> | ||
+ | <td>pET-3A DNA</td> <td> 50.0</td></tr> | ||
+ | <tr> | ||
+ | <td>Restriction enzyme</td> <td> 1.0</td></tr> | ||
+ | <tr> | ||
+ | <td>Total</td> <td>20.0</td></tr></table> | ||
+ | |||
+ | <li type="disc">Please note that 50uL of plasmid DNA was added to this reaction because we only obtained a low yield from the Invitrogen purification kit.</li> | ||
+ | |||
+ | |||
+ | |||
+ | <h3> Figure 13. Restriction enzyme digest on Qiagen kit results: </h3> | ||
+ | |||
+ | |||
+ | <p><b>Figure 13.</b> GelRed post-stained 1% agarose gel of restriction enzyme digested with BamH1 and Nde1 of pET-3A plasmid purified with the Qiagen kits. Digest has been successful and shows that the purified plasmids have been linearized because they show a band of expected size and intensity.</p> | ||
+ | |||
+ | |||
+ | <h3> Figure 14. Restriction enzyme digest on Invitrogen kit results: </h3> | ||
+ | |||
+ | <p><b>Figure 14.</b> GelRed post-stained 1% agarose gel of restriction enzyme digested with BamH1 and Nde1 of pET-3A plasmid purified with the Invitrogen kit. The samples have been pooled with this kit because there wasn’t much plasmid recovered. The enzyme digest was <u>successful.</u></p> | ||
+ | |||
+ | <hr> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <p></p><p> | ||
+ | <big> | ||
+ | |||
+ | <b> | ||
+ | |||
+ | 3rd October 2010 <p> | ||
+ | |||
+ | Plasmid Prep using Promega Wizard® Plus SV Minipreprs DNA Purification System<p> </big> </font> </b> | ||
+ | |||
+ | |||
+ | <li type="disc">The pET-3A plasmid was purified using the Promega Wizard® Plus SV Minipreps DNA Purification System as per the manufacturer’s protocols</li> | ||
+ | |||
+ | <li type="disc">The only difference made to the protocol was that 50uL of Gibco H20 was added instead of 100uL for the elution step. This was labeled ‘F1’</li> | ||
+ | |||
+ | <li type="disc">Then 95uL Gibco H2O was added and collected. This was labeled ‘F2’ | ||
+ | </li> | ||
+ | <li type="disc">To try and obtain a higher yield the cell lysate was divided into 10 smaller tubes and a small column was used</li> | ||
+ | |||
+ | <li type="disc">The purification was run on a GelRed stained 1.5% agarose gel and photo taken for visualization (see below).</li> | ||
+ | |||
+ | |||
+ | <h3> Figure 15. Promega Wizard® Plus SV Minipreps DNA Purification System results: </h3> | ||
+ | |||
+ | |||
+ | <p><b> Figure 15.</b> GelRed post-stained 1.5% agarose gel of pET-3A plasmid purification using Promega Wizard® Plus SV Minipreps DNA Purification System. In lanes 1 and 10 there is a 1kb Molecular ladder. In lanes 2-10 there is the 10 pooled cell lysate samples ‘F1’ that have been collected after 50uL of Gibco H20 was added. There is a band visible in each row from the first set of eluted samples. </p> | ||
+ | |||
+ | |||
+ | |||
+ | <h3>Figure 16. </h3> | ||
+ | |||
+ | |||
+ | <p><b>Figure 16. GelRed post-stained 1.5% agarose gel of pET-3A plasmid purification using Promega Wizard® Plus SV Minipreps DNA Purification System. In lanes 1 and 10 there is a 1kb Molecular ladder. In lanes 2-10 there is the 10 pooled cell lysate samples ‘F2’ that have been collected after additional 95uL of Gibco H2O was added. There is a feint band visible in each row from the second set of eluted samples. </b></p> | ||
+ | |||
+ | <hr><p></p><p> | ||
+ | <big> | ||
+ | |||
+ | <b> | ||
+ | |||
+ | 3rd October 2010 <p> | ||
+ | |||
+ | Restriction enzyme digest of purified pET-3A vector<p> </big> </font> </b> | ||
+ | <p> | ||
+ | <li type="disc">The enzyme digest was again performed on the pET-3A vector that had been purified with the Invitrogen Midi Prep kit (performed on the 31st September)</li> | ||
+ | <li type="disc">The enzyme digest was also performed on the pET-3A vector that had been purified with the Wizard® Plus SV Minipreps (performed on 3rd September)</li> | ||
+ | <li type="disc">The pET-3A vector was digested with BamH1 and Nde1 restriction enzymes so that the D. radiodurans and A. tumefaciens bacteriophytochrome-RBS-HO site operons that we have constructed can be ligated into the pET-3A vector | ||
+ | </li><li type="disc">The digests were run on a GelRed stained 1.5% agarose gel and photo taken for visualization (see below).</li> | ||
+ | |||
+ | |||
+ | </p><p><li type="disc">The reaction mastermix for the restriction digest to be performed on the Invitrogen Midi Prep kit purified plasmid was set up as per the following recipe (per sample): | ||
+ | |||
+ | Mastermix:</th> <th>Amount per sample (uL)</th></tr> | ||
+ | <tr> | ||
+ | <td> Gibco H2O</td> <td> 2.5</td> </tr> | ||
+ | <tr> | ||
+ | <td> 10x Buffer</td> <td>6.0</td></tr> | ||
+ | <tr> | ||
+ | <td> BSA (10ug/uL)</td> <td> 0.5</td></tr> | ||
+ | <tr> | ||
+ | <td>pET-3A DNA</td> <td> 50.0</td></tr> | ||
+ | <tr> | ||
+ | <td>Restriction enzyme</td> <td> 1.0</td></tr> | ||
+ | <tr> | ||
+ | <td>Total</td> <td>20.0</td></tr></table> | ||
+ | |||
+ | |||
+ | <li type="disc">Please note that 50uL of plasmid DNA was added to this reaction because we only obtained a low yield from the Invitrogen purification kit.</li> | ||
+ | |||
+ | |||
+ | <h3>Figure 17. </h3> | ||
+ | |||
+ | |||
+ | <p><b>Figure 17. </b> GelRed post-stained 1.5% agarose gel of BamH1 and Nde1 restriction enzyme digested pET-3A purified using Wizard® Plus SV kit. In lane 1 and 13 there is a 1kb ladder. In lanes 2-6 there is the BamH1 digest. In lane 6 there is a gap. In lanes 7-11 there is the Nde1 digest. Digest has been successful and shows that it is plasmid DNA not genomic DNA that has been purified. </p> | ||
+ | |||
+ | |||
+ | <h3>Figure 18</h3> | ||
+ | |||
+ | <p><b>Figure 18. </b>GelRed post-stained 1.5% agarose gel of BamH1 and Nde1 restriction enzyme digested pET-3A purified using Invitrogen kit. In lane 5 and 9 there is a 1kb ladder. In lane 6 there is the BamH1 digest. In lane 7 there is a gap. In lanes 8 there is the Nde1 digest. Digest has been successful and shows that it is plasmid DNA not genomic DNA that has been purified.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <hr><p></p><p> | ||
+ | <big> | ||
+ | |||
+ | <b> | ||
+ | |||
+ | 6th October 2010 <p> | ||
+ | |||
+ | Restriction enzyme digest of purified pET-3A vector obtained from various purification kits<p> </big> </font> </b> | ||
+ | <p> | ||
+ | <li type="disc">As we needed approximately 200ng of plasmid for cloning the plasmid that had been with purified with various kits was pooled </li> | ||
+ | <li type="disc">The pooled purified plasmids were then digested with BamH1 and Nde1 restriction enzymes</li> | ||
+ | <li type="disc">The digests were run on a GelRed stained 1.5% agarose gel and photo taken for visualization (see below)</li> | ||
+ | <p><li type="disc">The reaction mastermix for the restriction digest to be performed on the Invitrogen Midi Prep kit purified plasmid was set up as per the following recipe (per sample): </li> | ||
+ | |||
+ | |||
+ | Mastermix:</th> <th>Amount per sample (uL)</th></tr> | ||
+ | <tr> | ||
+ | <td> Gibco H2O</td> <td> 3.0</td> </tr> | ||
+ | <tr> | ||
+ | <td> 10x Buffer</td> <td>4.9</td></tr> | ||
+ | <tr> | ||
+ | <td> BSA (10ug/uL)</td> <td> 1.0</td></tr> | ||
+ | <tr> | ||
+ | <td>pET-3A DNA</td> <td> 30.0</td></tr> | ||
+ | <tr> | ||
+ | <td>Restriction enzyme</td> <td> 2.0</td></tr> | ||
+ | <tr> | ||
+ | <td>Total</td> <td>20.0</td></tr></table> | ||
+ | |||
+ | |||
+ | <h3> Figure 19 </h3> | ||
+ | |||
+ | |||
+ | <p><b>Figure 19</b>. GelRed post-stained 1.5% agarose gel of BamH1 and Nde1 restriction enzyme digested pET-3A purified using pooled samples purified with various kits. In lane 1 and 4 and 7 there is a 1kb ladder. In lane 2 there is the pooled Wizard product digested with BamH1. In lane 3 there is the pooled Qiagen product digested with BamH1. In lane 5 there is the pooled Wizard product digested with Nde1. In lane 6 there is the pooled Qiagen product digested with Nde1. Digest has been successful and shows that it is plasmid DNA not genomic DNA that has been purified. </p> | ||
+ | |||
+ | |||
+ | |||
+ | <hr><p></p><p> | ||
+ | <big> | ||
+ | |||
+ | <b> | ||
+ | |||
+ | 6th October 2010 <p> | ||
+ | |||
+ | Ligation and plasmid clean up prior to cloning<p> </big> </font> </b> | ||
+ | <p> | ||
+ | <li type="disc">The gene products were ligated into the pET-3A vector using the Promega T4 ligation system | ||
+ | </li> | ||
+ | <li type="disc">After the restriction enzyme digest the products had to be cleaned of all excess salts and buffers | ||
+ | </li> | ||
+ | |||
+ | |||
+ | <hr><p></p><p> | ||
+ | <big> | ||
+ | |||
+ | <b> | ||
+ | |||
+ | 15th October 2010 <p> | ||
+ | Transformation into BL21(DE3) and Top 10 cells for pGEM-T Easy<p> </big> </font> </b> | ||
+ | |||
+ | <li type="disc">The final products were transformed into two strains of E. coli | ||
+ | </li><li type="disc">The first strain BL21(DE3) were used so that the HO gene could be expressed, if the transformation is successful green E. coli colonies will be observed | ||
+ | </li><li type="disc">The top 10 cells were also transformed using blue/white screening methods as the pGEM-T Easy storage vector contains the lacZ gene. | ||
+ | </li><li type="disc">Unfortunately none of the BL21(DE3) expressed the HO gene as all colonies grown were not green | ||
+ | </li><li type="disc">The blue/white screening of the pGEM-T Easy clones were successful as there were a few clones that were white in colour (showing that the transformation had worked in these clones) | ||
+ | </li> | ||
Revision as of 10:08, 24 October 2010
PROJECT LAB BOOK
Welcome to the Macquarie University project lab book page!
DAY-BY-DAY PROGRESS FOR THE TRANSFORMATION AND CLONING
22nd September 2010
Plasmid Prep using Qiagen Plasmid Midi Prep Kit
Figure 10. Qiagen Plasmid Midi Prep results:
Figure 10. GelRed post-stained 1.5% agarose gel of pET-3A Plasmid purification. In lanes 1, 4, 5 and 15 there is a 1kb Molecular ladder. In lane 2 there is the Plasmid purification sample A and in lane 3 is the Plasmid purification sample B. In lanes 6 to 14 there are flow-throughs that were collected at different stages throughout the protocol. There is some DNA visible in these lanes so some of the DNA has been lost. Possibly genomic DNA they are quite high (didn’t move through the wells)
Nanodrop results:
Sample | Concentration | A260/280 ratio |
---|---|---|
A | 36.6ng/uL | 1.71 |
B | 35.0ng/uL | 1.73 |
Looking at these results it seems that we have approximately 100uL of 36.6ng/uL plasmid sample A and approximately 100uL of 35.0ng/uL plasmid sample B.
A restriction digest will confirm that it is plasmid DNA not genomic DNA.
27th September 2010
Plasmid Prep using Qiagen Plasmid Midi Prep Kit (2nd attempt)
Figure 11. Qiagen Plasmid Midi Prep results (2nd attempt)
Figure 11. GelRed post-stained 1.5% agarose gel of pET-3A Plasmid purification. In lanes 1 and 10 there is a 1kb Molecular ladder. In lanes 2-9 there is the pooled plasmid purification samples that have been collected in separate tubes. All samples show a band indicative of linearized plasmid except lane 7 – this sample has been mistreated in the purification process.
Figure 12. Qiagen Plasmid Midi Prep results (2nd attempt)
Figure 12. GelRed post-stained 1.5% agarose gel of pET-3A Plasmid flow through collections. In lanes 1 and 9 there is a 1kb Molecular ladder. In lanes 2 – 9 there are the column flow throughs that were collected at different points throughout the protocol. In lanes 2 and 3 there is a feint band visible but this is expected as it is sample collected before the addition of sample to the column.
31st September 2010
Plasmid Prep using Invitrogen Midi Prep Kit
Figure 13. Plasmid Prep using Invitrogen Midi Prep Kit results
Figure 13. GelRed post-stained 1.5% agarose gel of pET-3A Plasmid purification using Invitrogen Midi Prep kit. This kit shows that we have recovered less plasmid but it is more highly purified than what was purified with the other kits because the bands are less smeared.
31st September 2010
Restriction enzyme digest on Purified pET-3A
Restriction enzyme recognition sites:
Restriction enzyme | Recognition site |
---|---|
BamH1 | 5’-CA (cut) TATG – 3’ and 3’-CCTAG (cut) G-5’ |
Nde1 | 5’-CA(cut)TATG-3’and 3’-GTAT(cut)AG-5’ |
Mastermix: | Amount per sample (uL) |
---|---|
Gibco H2O | 7.5 |
10x Buffer | 10.0 |
BSA (10ug/uL) | 0.5 |
pET-3A DNA | 1.0 |
Restriction enzyme | 1.0 |
Total | 20.0 |
Mastermix: | Amount per sample (uL) |
---|---|
Gibco H2O | 2.5 |
10x Buffer | 6.0 |
BSA (10ug/uL) | 0.5 |
pET-3A DNA | 50.0 |
Restriction enzyme | 1.0 |
Total | 20.0 |
Figure 13. Restriction enzyme digest on Qiagen kit results:
Figure 13. GelRed post-stained 1% agarose gel of restriction enzyme digested with BamH1 and Nde1 of pET-3A plasmid purified with the Qiagen kits. Digest has been successful and shows that the purified plasmids have been linearized because they show a band of expected size and intensity.
Figure 14. Restriction enzyme digest on Invitrogen kit results:
Figure 14. GelRed post-stained 1% agarose gel of restriction enzyme digested with BamH1 and Nde1 of pET-3A plasmid purified with the Invitrogen kit. The samples have been pooled with this kit because there wasn’t much plasmid recovered. The enzyme digest was successful.
3rd October 2010
Plasmid Prep using Promega Wizard® Plus SV Minipreprs DNA Purification System
Figure 15. Promega Wizard® Plus SV Minipreps DNA Purification System results:
Figure 15. GelRed post-stained 1.5% agarose gel of pET-3A plasmid purification using Promega Wizard® Plus SV Minipreps DNA Purification System. In lanes 1 and 10 there is a 1kb Molecular ladder. In lanes 2-10 there is the 10 pooled cell lysate samples ‘F1’ that have been collected after 50uL of Gibco H20 was added. There is a band visible in each row from the first set of eluted samples.
Figure 16.
Figure 16. GelRed post-stained 1.5% agarose gel of pET-3A plasmid purification using Promega Wizard® Plus SV Minipreps DNA Purification System. In lanes 1 and 10 there is a 1kb Molecular ladder. In lanes 2-10 there is the 10 pooled cell lysate samples ‘F2’ that have been collected after additional 95uL of Gibco H2O was added. There is a feint band visible in each row from the second set of eluted samples.
3rd October 2010
Restriction enzyme digest of purified pET-3A vector
Figure 17.
Figure 17. GelRed post-stained 1.5% agarose gel of BamH1 and Nde1 restriction enzyme digested pET-3A purified using Wizard® Plus SV kit. In lane 1 and 13 there is a 1kb ladder. In lanes 2-6 there is the BamH1 digest. In lane 6 there is a gap. In lanes 7-11 there is the Nde1 digest. Digest has been successful and shows that it is plasmid DNA not genomic DNA that has been purified.
Figure 18
Figure 18. GelRed post-stained 1.5% agarose gel of BamH1 and Nde1 restriction enzyme digested pET-3A purified using Invitrogen kit. In lane 5 and 9 there is a 1kb ladder. In lane 6 there is the BamH1 digest. In lane 7 there is a gap. In lanes 8 there is the Nde1 digest. Digest has been successful and shows that it is plasmid DNA not genomic DNA that has been purified.
6th October 2010
Restriction enzyme digest of purified pET-3A vector obtained from various purification kits
Figure 19
Figure 19. GelRed post-stained 1.5% agarose gel of BamH1 and Nde1 restriction enzyme digested pET-3A purified using pooled samples purified with various kits. In lane 1 and 4 and 7 there is a 1kb ladder. In lane 2 there is the pooled Wizard product digested with BamH1. In lane 3 there is the pooled Qiagen product digested with BamH1. In lane 5 there is the pooled Wizard product digested with Nde1. In lane 6 there is the pooled Qiagen product digested with Nde1. Digest has been successful and shows that it is plasmid DNA not genomic DNA that has been purified.
6th October 2010
Ligation and plasmid clean up prior to cloning
15th October 2010
Transformation into BL21(DE3) and Top 10 cells for pGEM-T Easy