Team:Lethbridge CCS/Notebook/October

For earlier work, see September Notebook, or go back to Main CCS Notebook.

4 Oct 2008
(Marc, Glenda, Nathan)

Met to discuss why we had not obtained green colonies from our transformation as expected. There was definitely cutting happening from the EcoRV digest, but the bands showed up at the wrong size. Final conclusion was that probably nothing had worked right from step 1! Most likely what we thought was pSB1A7 actually wasn't, a conclusion we came to based on our own funny results, and on some of the results obtained by the U of L team. So we've probably just been seeing the original template plasmid in all our follow up gels, etc. Decided to start again, this time using pSB1A2 (though it doesn't have the double terminators we would actually like to see), as the U of L team has it and knows it works. So new primers were designed for this, as well as for xylE, a gene the ULeth team is using, which we hope to use to see whether we can really build a 'new' BioBrick using our method.

6 Oct 2008
(Peter, Elizabeth, Glenda, Nathan)

Reviewed Saturday's discussion; checked that primers were correct and sent off order; discussed our logo and ordering of T-shirts; reviewed steps we would have to go through once our new primers arrive

11 Oct 2008
(Peter, Elizabeth, Marc, Glenda)


 * Roxanne had already resuspended and diluted our primers (psB1A2 forward and reverse; LIC_xylE forward and reverse) [100 &mu;M stock; 10&mu;M working].
 * Set up and ran PCRs on pSB1A2 and LIC_xylE
 * for each, we did 1 x, 1/10 x and 1/100 x of our template and a range of annealing temperatures (54&deg;C, 58&deg;C, 62&deg;C for pSB1A2; 48&deg;C, 52&deg;C, 56&deg;C for LIC_xylE), giving nine variations for each PCR;
 * used materials and amounts as per Phusion polymerase kit

- Cycling temperatures and times for PCR Initial denaturation          98&deg;C                 30 sec    ___ Denaturation                  98&deg;C                 10 sec       | Annealing                     gradient as above    30 sec       |--- 30 cycles Extension                     72&deg;C                  1 min    ___| Final extension               72&deg;C                 10 min Hold                          4&deg;C                  hold

54&deg;C     58&deg;C      62&deg;C 1x     1         4         7 1/10x     2         5         8 1/100x     3         6         9 [lanes: 1kb ladder, 1-5, 1kb ladder, 6-9, blank, 1kb ladder]
 * numbering:
 * ran a 1% agarose gel on pSB1A2_LIC; expected size ~2000 bp. No results.  After discussion, we think this may be due to erroneously putting in only enough polymerase for one reaction, not 10.

[lanes: 1kb ladder, 1-5, 1kb ladder, 6-9, blank, 1kb ladder]
 * ran a 1% agarose gel on xylE_LIC; expected size ~1000bp.

13 Oct 2008
(Peter, Marc - yes, we were in the lab on Thanksgiving!)

[Lanes: 1kb ladder, 1-9, blank, 1kb ladder]
 * Re-did PCR of pSB1A2, this time using GFP complete (1) plasmid-prepped by Roxanne.


 * Seems to have worked. Possible problem: circular pSB1A2 with GFP complete (~3000bp) may run at the same place as linear pSB1A2 alone (~2000bp). Will run another gel tomorrow to check.

14 Oct 2008
(Marc)

[Lanes: 1kb ladder, GFP complete (1), #2, #5, #8, 1kb ladder]
 * Ran 1% agarose gel to check where pSB1A2 with GFP complete (template for our pSB1A2 PCR) runs on a gel.


 * Unfortunately, template (circular) runs at the same place as our PCR product. We could possibly digest the BioBrick plasmid first; then at least there wouldn't be any competition for transformation.

(Elizabeth, Peter, Glenda)

1 &mu;L PCR product 2 &mu;L 5x buffer             incubated for 1 hour at room temperature 1 &mu;L ligase 6 &mu;L Water
 * Used Fermentas Rapid Ligation kit on our around the world PCR products(1/10, 1/100 concentration, 54&deg;)


 * Did a transformation of ligated DNA into highly competent cells (using 1 &mu;L ligation mix (1/10, 1/100, pUC19 control))
 * left on ice for 30 min
 * heat-shock for 30 sec at 42 degrees
 * ice for 2 minutes
 * add 900 &mu;L SOC medium, 1 hour in shaker at 37 degrees
 * plated concentrated and dilute of each of above dilutions
 * incubated overnight


 * At the U of L iGEM meeting, we discussed our concern about template running at same place as PCR product. HJ recommended doing a DpnI digest since this would cleave the template but leave our PCR product intact. We could then run a gel to compare sizes.

15 Oct 2008
(Glenda, Elizabeth, Peter)


 * Interestingly enough, where we had expected to see all or almost all green colonies (if template was present as expected), but in fact there were only white colonies
 * Grew up colonies (2 each from our 1/10 and 1/100 dilute plates)
 * 5 &mu;L ampicillin, add with colonies to culture
 * Incubate at 37&deg;C for 12 hours


 * Set up DpnI digestion

15 &mu;L PCR product/template (used 1x LIC_GFP) 1.75 &mu;L 10x Tango buffer 1 &mu;L DpnI

Lane 1: 1 kb GeneRuler ladder Lane 2: GFP with DpnI   Lane 3: GFP without DpnI    Lane 4: LIC_GFP with DpnI    Lane 5: LIC_GFP without DpnI    Lane 6: 1 kb GeneRuler ladder
 * Incubated 1 hour at 37&deg;C and then ran a 1% agarose gel
 * gel looked a little strange (not well-defined), but seems to verify that our early amplification worked; Lane 2 had marks at a variety of lengths, indicating cutting of GFP with DpnI, while the other lanes all looked very similar,with only one length, indicating there was very little template left in our LIC_GFP. The only slightly puzzling thing was the length of the DNA it seemed to show (shorter than expected)

16 Oct 2008
(Elizabeth, Marc)


 * Performed miniprep of pSB1A2_LIC from 'LIC_GFP' cultures, as per QIAprep Spin Kit.
 * Notes: did not do optional PB buffer step; pellet of 1/100 #2 was very thin and elongated.


 * Prepared glycerol stocks of all four cultures. Stored in HJ's -80&deg;C freezer.


 * Quantified resulting plasmid concentrations using UV/vis spectrophotometer at 1:25 dilution.

Sample    Concentration (ng/&mu;L)     260/280nm absorbance ratio 1/10 #1              57                      1.83  1/10 #2               81                      1.83 1/100 #1               88                      1.95 1/100 #2               50                      1.90

[Lanes: 1/25 dilution of 1/10 #1, 1kb ladder (punctured well?), 1/10 #1, 1/10 #2, 1/100 #1, 1/100 #2, 1kb ladder, 1/25 dilution of 1/10 #2]
 * Ran 1% agarose gel to check size of purified plasmid (5 &mu;L loading volumes; 100V for 25min)


 * All plasmid preps seem to have worked; circular plasmid is running a little below 2000bp, which seems a little small but is probably OK. May be due to insufficiently resolved gel.

17 Oct 2008
(Glenda, Elizabeth, Peter)

- components and amounts (for each of 4 colonies grown up previously) DNA     5 &mu;L Buffer  1 &mu;L           incubated overnight EcoRV   1 &mu;L Water   3 &mu;L
 * Set up EcoRV digest of purified pSB1A2_LIC plasmid (Fermentas)

18 Oct 2008
(Paul, Peter, Elizabeth, Marc)

[Lanes: 1kb ladder, 1/10 #1 Cut & Uncut, 1/10 #2 Cut & Uncut, 1/100 #1 Cut & Uncut, 1/100 #2 Cut & Uncut, 1kb ladder]
 * Ran 1% agarose gel to check results of yesterday's EcoRV digest. (2 &mu;L loading volumes; 5 &mu;L ladder; 100V for 35 min)
 * Some strange results, but there was definitely digestion. It seems that 1/10 #1 may not have been fully digested; no idea why the uncut 1/100 #1 didn't show up.   The #2 samples, which look 'normal', will be used for subsequent steps.
 * NB: uncut plasmid is around 1700bp, which is a little smaller than it looked on the previous gel. Cut (linear) plasmid is right around 2000bp where it belongs.

Vector                                Buffer dCTP          3.33 &mu;L                 dCTP                 3.33 &mu;L T4 Buffer     2    &mu;L                 T4 Buffer            2    &mu;L pSB1A2_LIC    2.5  &mu;L                 xylE_LIC or GFP_LIC  2.5  &mu;L T4 Polymerase 0.4  &mu;L                 T4 Polymerase        0.4  &mu;L Water         1.77 &mu;L                 Water                1.77 &mu;L
 * Performed T4 DNA polymerase digestions on EcoRV-cut pSB1A2_LIC, and on PCR-amplified xylE_LIC and GFP_LIC.

Vector                                Buffer dCTP          3.33 &mu;L                 dCTP                 3.33 &mu;L NEBuffer2     1    &mu;L                 NEBuffer2            1    &mu;L pSB1A2_LIC    2.5  &mu;L                 GFP_LIC              2.5  &mu;L Klenow        2    &mu;L                 Klenow               2    &mu;L Water         1.17 &mu;L                 Water                1.17 &mu;L
 * Performed Klenow fragment digestions on EcoRV-cut pSB1A2_LIC, and on PCR-amplified GFP_LIC.
 * For both T4 &amp; Klenow treatments, let digestion run for 20 minutes at 37&deg;C.


 * Purified T4- &amp; Klenow-digested DNA with Qiagen MinElute PCR purification kit.
 * Checked concentrations of T4- &amp; Klenow-treated DNA samples using UV/vis spectrophotometer (1:50 dilution)

Sample            Concentration (ng/&mu;L)     260/280nm absorbance ratio T4 pSB1A2_LIC 10            41                        1.29 T4 pSB1A2_LIC 100           69                        1.07 T4 xylE_LIC                 75                        1.74 T4 GFP_LIC                  66                        1.81 Klenow pSB1A2_LIC           47                        1.27 Klenow GFP_LIC             243                        1.53      &mdash; concentration very high - not sure why


 * Stored purified plasmids &amp; inserts in freezer overnight.

20 Oct 2008
(Peter, Elizabeth, Glenda, Marc) 1 &mu;L T4-treated pSB1A2_LIC 100 + 3 &mu;L T4-treated GFP_LIC 1 &mu;L T4-treated pSB1A2_LIC 10 + 2 &mu;L T4-treated xylE_LIC 1 &mu;L Klenow-treated pSB1A2_LIC + 3 &mu;L Klenow-treated GFP_LIC
 * Mixed T4- and Klenow-treated vectors with corresponding inserts (10 min at room temp.) to allow LIC overhangs to interact, aiming for approximately 3:1 insert:vector molar ratio.
 * Transformation of LIC-treated vectors:
 * Added each set of mixed vectors &amp; inserts to 25 &mu;L DH5&alpha; MaxEfficiency chemically competent cells, and left on ice for 30 minutes.
 * 30-second heat-shock at 42&deg;C
 * 2 minutes on ice
 * added 900 &mu;L SOC media to each tube, and left in shaker at 37&deg;C for one hour


 * Plated 100 &mu;L of each culture on LB+Amp plates ('dilute')
 * Pelleted remaining cells, removed 800 &mu;L media, and plated remaining 100 &mu;L culture on LB+amp plates ('concentrated')

21 Oct 2008
(Peter)


 * Checked plates from LIC transformation.

Transformation                    No. of colonies T4 GFP_LIC    - dilute                    6 - concentrated           ~30 T4 xylE_LIC   - dilute                 ~100 - concentrated          ~500 Klenow GFP_LIC - dilute                   1 - concentrated           ~15


 * Transformation seems to have worked, since colonies were observed. None of them were green, however, so the LIC procedure didn't work.  Unfortunately, we don't have time to carry out more trials, so we are not sure why not.

27 Oct 2008

 * Sent off pSB_LIC part to the Registry

28 Oct 2008
(all)


 * Met with U of L team to practice Jamboree presentations, and to provide &amp; receive feedback.

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